Rabies virus vector systems and compositions and methods thereof

ABSTRACT

Rabies Virus compositions and methods are provided. The full-length sequence of Rabies Virus strain Evelyn-Rokitnicki-Abelseth (ERA) is disclosed. A reverse genetics system for producing recombinant ERA virus and derivatives thereof is provided, along with compositions including ERA and/or ERA derivative strain viruses, nucleic acids and/or proteins. In some instances, the compositions are immunogenic compositions useful for the pre- or post-exposure treatment of Rabies Virus.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a divisional of U.S. application Ser. No. 12/090,083, filed Apr.11, 2008, issued as U.S. Pat. No. 7,863,041 on Jan. 4, 2011, which isthe U.S. National Stage of International Application No.PCT/US2006/040134, filed Oct. 13, 2006, which was published in Englishunder PCT Article 21(2), and which claims the benefit of U.S.Provisional Application No. 60/727,038, filed Oct. 14, 2005. All of theabove-referenced applications are incorporated herein by reference intheir entirety.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made by the Centers for Disease Control andPrevention, an agency of the United States Government. Therefore, theUnited States Government has certain rights in this invention.

FIELD

This disclosure relates to the field of virology. More specifically,this disclosure relates to compositions and methods that are useful forthe production of immunogenic compositions for protecting mammals frominfection by rabies virus.

BACKGROUND OF THE DISCLOSURE

Rabies remains one of the most dreadful infectious diseases affectinghuman and animals, despite significant scientific advances in itsprevention and control. Rabies presents as a distinct problem indifferent parts of the world. In the Americas, reservoirs of rabiesexist in many wild animal species, including raccoons, skunks, foxes,and bats (Rupprecht et al., Emerg. Infect. Dis. 1(4):107-114, 1995).Outbreaks of rabies infections in these terrestrial mammals are found inbroad geographic areas across the United States. For example, raccoonrabies affects an area of more than 1 million square kilometers fromFlorida to Maine. Although wildlife rabies still exists in developedcountries, progress has been made in control and elimination of wildliferabies using oral immunization of wild animals.

Nonetheless, rabies remains a major threat to public health and persiststo cause between 50,000 and 60,000 human deaths each year (World HealthOrganization, April 2003). Humans get infected with the rabies virusmostly through bites from rabid domestic and wildlife animals. Indeveloping countries, dogs are responsible for about 94% of human rabiesdeaths. Dog rabies is still epizootic in most countries of Africa, Asiaand South America and in these countries dogs are responsible for mosthuman deaths from the disease. Controlling rabies virus infection indomestic and wildlife animals, therefore, not only reduces the mortalityin these animals but also reduces the risks of human exposure.

The rabies virus is transmitted through broken skin by the bite orscratch of an infected animal. Exposure to rabies virus results in itspenetration of peripheral, unmyelineated nerve endings, followed byspreading through retrograde axonal transport, replication occurringexclusively in the neurons, and finally arrival in the central nervoussystem (CNS). Infection of the CNS causes cellular dysfunction and death(Rupprecht & Dietzschold, Lab Invest. 57:603, 1987). Since rabies virusspreads directly from cell to cell, it largely evades immune recognition(Clark & Prabhakar, Rabies, In: Olson et al., eds., ComparativePathology of Viral Disease, 2:165, Boca Raton, Fla., CRC Press, 1985).

The rabies virus (RV) is a rhabdovirus—a nonsegmented RNA virus withnegative sense polarity. Within the Rhabdoviridae family, rabies virusis the prototype of the Lyssavirus genus. RV is composed of two majorstructural components: a nucleocapsid or ribonucleoprotein (RNP), and anenvelope in the form of a bilayer membrane surrounding the RNP core. Theinfectious component of all rhabdoviruses is the RNP core, whichconsists of the negative strand RNA genome encapsidated by nucleoprotein(N) in combination with RNA-dependent RNA-polymerase (L) andphosphoprotein (P). The membrane surrounding the RNP contains twoproteins: the trans-membrane glycoprotein (G) and the matrix (M)protein, located at the inner site of the membrane. Thus, the viralgenome codes for these five proteins: the three proteins in the RNP (N,L and P), the matrix protein (M), and the glycoprotein (G).

The molecular determinants of pathogenicity of various rabies virusstrains have not been fully elucidated. RV pathogenicity was attributedto multigenic events (Yamada et al., Microbiol. Immunol. 50:25-32,2006). For example, some positions in the RV genome if mutated, affectviral transcription or replication, reducing virulence. Mutations atserine residue 389 of the phosphorylation site in the N gene (Wu et al.,J. Virol. 76:4153-4161, 2002) or GDN core sequence of the highlyconserved C motif in the L gene (Schnell and Conzelmann, Virol.214:522-530, 1995) dramatically reduced RV transcription andreplication.

The G protein, also referred to as spike protein, is involved in cellattachment and membrane fusion of RV. The amino acid region at position330 to 340 (referred to as antigenic site III) of the G protein has beenidentified as important for virulence of certain strains of RV. Severalstudies support the concept that the pathogenicity of fixed RV strainsis determined by the presence of arginine or lysine at amino acidresidue 333 of the glycoprotein (Dietzschold et al., Proc. Natl. Acad.Sci. USA 80: 70-74, 1983; Tuffereau et al., Virol. 172: 206-212, 1989).

This phenomenon seems to apply at least to fixed rabies viruses such asCVS, ERA, PV, SAD-B19 and HEP-Flury strains (Anilionis et al., Nature294:275-278, 1981; Morimoto et al., Virol. 173:465-477, 1989). Forexample, rabies vaccine viruses possessing an amino acid differing fromArg at position 333 of the glycoprotein are described, for instance, inWO 00/32755 (describing RV mutants in which all three nucleotides in theG protein Arg₃₃₃ codon are altered compared to the parent virus, suchthat the Arg at position 333 is substituted with another amino acid);European Patent 350398 (describing an avirulent RV mutant SAG1 derivedfrom the Bern SAD strain of RV, in which the Arg at position 333 of theglycoprotein has been substituted to Ser); and European patentapplication 583998 (describing an attenuated RV mutant, SAG2, in whichthe Arg at position 333 in the G protein has been substituted by Glu).

Other strains, such as the RC-HL strain, possess an arginine residue atposition 333 of the G, but does not cause lethal infection in adult mice(Ito et al., Microbiol. Immunol. 38:479-482, 1994; Ito et al., J. Virol.75:9121-9128, 2001). As such, the entire G may contribute to thevirulence of RV, although the determinants or regions have notpreviously been identified. The G gene encodes the only protein thatinduces viral neutralizing antibody. At least three states of RVglycoprotein are known: the native state (N) being responsible forreceptor binding; an active hydrophobic state (A) necessary in theinitial step in membrane fusion process (Gaudin, J. Cell Biol.150:601-612, 2000), and a fusion inactive conformation (I). Correctfolding and maturation of the G play important roles for immunerecognition. The three potential glycosylated positions in ERA Gextracellular domain occur at Asn³⁷, Asn²⁴⁷ and Asn³¹⁹residues (Wojczyket al., Glycobiology. 8: 121-130, 1998), respectively. Nonglycosylationof G not only affects conformation, but also inhibits presentation ofthe protein at the cell surface. Thus, elucidating the moleculardeterminants underlying pathogenicity of rabies virus presents a complexproblem.

SUMMARY OF THE DISCLOSURE

The complete sequence of the virus strain corresponding to the fixedvaccine of Evelyn-Rokitnicki-Abelseth (ERA) for rabies virus isdisclosed herein, along with methods for sequencing this and otherstrains of lyssavirus.

A reverse genetics system for rabies virus is also described, inparticular using the rabies virus strain ERA as an exemplar. Use of a T7RNA polymerase, containing an eight amino acid nuclear localizationsignal (NLS) at the N terminal end facilitated virus recovery. Besidesthe parental ERA virus strain, several other derivative viruses aredescribed, including ERA- (deletion of the psi-region), ERAgreen1 (greenfluorescent protein gene inserted in psi region), ERAgreen2 (greenfluorescent protein gene inserted at the phosphoprotein and matrixprotein intergenic region), ERA2g (containing an extra copy of theglycoprotein in the psi-region), ERAg3 (with a mutation at amino acid333 in glycoprotein), ERA2g3 (with an extra copy of altered glycoproteinat amino acid 333 in psi-region), ERA-G (from which the glycoprotein hasbeen deleted) ERAgm (M and G genes switched in the genome), and ERAgmg(two copies of G in the rearranged ERAgm construct). The extratranscription unit was incorporated into ERA virus genome for efficientexpression of Open Reading Frames (ORFs). By optimizing propagationconditions, which are described herein, rescued viruses reach titers inexcess of 10⁹ ffu/ml in either bioreactors or stationary tissue flasks.

Also disclosed is a modified cell line that constitutively expresses theERA glycoprotein. The cell line, designated BSR-G, is useful for theproduction of recombinant, including attenuated and/or replicationdeficient, rabies virus.

The foregoing and other objects, features, and advantages of theinvention will become more apparent from the following detaileddescription.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A. Schematic illustration of the ERA transcription plasmid.Positions of the hammerhead ribozymes and antigenomic ERA genome areindicated graphically. Relative positions of the N, P, M G and Lproteins are shown in a 5′ to 3′ direction.

FIG. 1B. Schematic diagram of the construction of the full-length ERArabies virus genomic cDNA plasmid pTMF. RT-PCR products F1, F2fragments, and restriction enzyme recognition sites (Nhe1, Kpn1, Blp1,Pst1 and Not1) (not drawn to scale). The bar on the left indicates aRdRz-hammerhead ribozyme and the right bar indicates the HDVRz-hepatitisdelta virus ribozyme. The symbol ♦ indicates that Kpn1 or Pst1 siteswere deleted, and ⇓ vertical arrow indicates that Nhe1 or Not1 siteswere left intact.

FIG. 2. Schematic illustration of the proposed mechanism of NLST7 RNApolymerase autogene action by pNLST7 plasmids. The DNA-transfectionreagent complex is taken into cells by endocytosis. The majority of theDNA released from lysosomes and endosomes is retained in the cellcytoplasm. A limited amount of plasmids are transferred to thenucleus: 1) through a CMV immediate early promoter, the NLST7 gene istranscribed by cellular RNA polymerase II; 2) mature NLST7 mRNA istransported from the nucleus to the cytoplasm for NLST7 RNA polymerasesynthesis; 3) Newly synthesized NLST7 RNA polymerase is trans-located tothe nucleus, while a trace amount of NLST7 remains in the cytoplasm; 4)NLST7 RNA polymerase initiates transcription through a pT7 promoter. Byposttranscriptional modifications, additional NLST7 mRNA is produced forprotein synthesis, thus increasing virus recovery efficiency.

FIG. 3. Schematic diagram of the ten derivative ERA virus genomes. Thesize of each gene is not drawn to scale. Symbol “*” denotes mutations ofG at the Aa333 residue and Ψ is the Psi-region.

FIG. 4A. Analysis of recovered ERA-G virus in transfected cells, spreadand growth in a BHK-G cell line. In A, the ERA-G viral foci wererestrained even after seven-day post transfection with plasmids forvirus recovery. In B, rescued ERA-G virus did not spread after passagein normal BSR cells. Only individual cells were stained by DFA. In C,the ERA-G virus grew well in the constitutive BHK-G cell line.

FIG. 4B. Analysis of G expression in a BHK-G cell line. By indirectfluorescent staining, the ERA rabies virus G was expressed in thecytoplasm in a stable cell line BHK-G.

FIG. 4C. Analysis of G mRNA in ERA-G virus-infected cells by Northernblot with a G probe. Lane 2 shows that the G gene mRNA was detected inERA-G virus infected BHK-G cells, while virus genomic RNA was not. Lane1 was the control of total RNA from ERA-rabies virus-infected BHK-Gcells, in which both G mRNA and viral genomic RNA were detected.

FIG. 5. Single-step virus growth curve. All recovered rabies virus ERAstrains grew to 10⁹ or 10¹⁰ ffu/ml, but the ERA-G only reached 10⁷ffu/ml.

FIG. 6. Green foci in ERAgreen1/ERAgreen2 rabies virus-infected BSRcells. Trans 1 is the transcriptional unit incorporated at the Psi and Lintergenic regions. Trans2 is the transcriptional unit at the P and Mintergenic region. Both ERAgreen2 and ERAgreen1 expressed the GFPprotein stably in virus-infected BSR cells, while the occurrence of thegreen foci of ERAgreen2 was 48 hours earlier after virus infection thanin ERAgreen1.

FIG. 7. Analysis of G mRNA expression in double G, and G, M rearrangedERA rabies viruses. In the Northern blot with a G probe, the measurementby density photometry of G mRNA in ERA2g (lane 1), ERAgm (lane 3) andERA2g3 (lane 4) represented enhanced mRNA levels, compared withERA-virus-infected cells (lane 2). The ratios were calculated by the useof ERA-virus as 100%.

FIG. 8A. Morbidity induced by in vivo inoculation with recombinant ERAand derivatives. Three-week old mice were inoculated intramuscularlywith the eight recovered viruses. At 10 days post inoculation, in ERA,ERA- and ERAgreen1 groups, 50%, 50% and 20% of mice showed compatibleclinical signs of rabies, but no mortality, respectively. No adversesigns were observed in the other groups.

FIG. 8B. Post challenge survival in mice inoculated with recombinant ERAand derivatives. Mice surviving from the tests shown in FIG. 8A werechallenged intramuscularly with a Texas dog/coyote rabies virus. At 5days post challenge, in the ERA and ERA-groups, 40 and 62% of miceshowed signs of rabies and were euthanized, respectively. In all othergroups, no signs of rabies were observed.

FIG. 8C. Survival following i.c. inoculation with recombinant ERA andERAg3 viruses. Three-week old mice were inoculated intracerebrally withERA and ERAg3 virus strains, respectively. All mice succumbed 15 dayspostinoculation in the ERA group, while in the ERAg3 group, all micesurvived with no clinical signs.

FIG. 8D. Survival following i.c. inoculation of suckling mice. Two-dayold suckling mice were inoculated intracerebrally with ERAg3 and ERA-Gvirus constructs, respectively. All mice succumbed in the ERAg3 group,while no mice died in the ERA-G group.

FIG. 8E. Neutralizing antibody titer in mice inoculated with recombinantERA and derivative viruses. Mouse neutralization antibody titers weredetermined by the RFFIT, ranging from 1.36 to 5.61 IU per ml among thevirus-inoculated groups.

FIG. 9A. Survival following infection with Alabama Bat Rabies virus.Hamsters were inoculated with live Alabama Bat Rabies Virus, thentreated post-exposure with either ERAg3 virus or with rabies immuneglobulin and commercially available inactivated RV vaccine. Survival wasassessed over a more than three month period.

FIG. 9B. Survival following infection with Thai Street Dog Rabies virus.Hamsters were inoculated with live Alabama Bat Rabies Virus, thentreated post-exposure with either ERAg3 virus or with rabies immuneglobulin and commercially available inactivated RV vaccine. Survival wasassessed over a more than three month period.

FIG. 9C. Survival following infection with Texas Coyote Dog Rabiesvirus. Hamsters were inoculated with live Alabama Bat Rabies Virus, thentreated post-exposure with either ERAg3 virus or with rabies immuneglobulin and commercially available inactivated RV vaccine. Survival wasassessed over a more than three month period.

SEQUENCE LISTING

The nucleic and amino acid sequences listed in the accompanying sequencelisting are shown using standard letter abbreviations for nucleotidebases, and three letter code for amino acids, as defined in 37 C.F.R.1.822. Only one strand of each nucleic acid sequence is shown, but thecomplementary strand is understood as included by any reference to thedisplayed strand unless the context makes it clear that only one strandis intended. As appropriate, it will be understood that a sequencepresented as DNA can be converted to RNA by replacing thiamine residueswith uracils. The Sequence Listing is submitted as an ASCII text file,created on Nov. 19, 2010, 285 KB, which is incorporated by referenceherein. In the accompanying sequence listing:

SEQ ID NO: 1. ERA CDC wild type virus, 11,931 nucleotides

1-58 nucleotides, Leader region

71-1420 nucleotides, N gene

1514-2404 nucleotides, P gene

2496-3101 nucleotides, M gene

3317-4888 nucleotides, G gene

4964-5362 nucleotides, Psi-region

5417-11797 nucleotides, L gene

11862-11931 nucleotides, Trailer region

SEQ ID NO: 2. ERACDC: 71 to 1420:450 aa, N protein.

SEQ ID NO: 3. ERACDC: 1514 to 2404:297 aa, P protein.

SEQ ID NO: 4. ERACDC: 2496 to 3101:202 aa, M protein.

SEQ ID NO: 5. ERACDC: 3317 to 4888:524 aa, G protein.

SEQ ID NO: 6. ERACDC: 5417 to 11797:2127 aa, L protein.

SEQ ID NO: 7. Recombinant ERA (rERA) recovered by reverse geneticssystem is 11,930 nucleotides. The specific poly (A₈) tract between Ggene and psi-region in wild type ERA strain was mutated to a poly (A₇)tract in recombinant ERA reverse genetics system as a sequence marker.In light of this, rERA is one nucleotide shorter than wild type ERA. Allthe other sequence information is exactly the same.

SEQ ID NO: 8. ERAg3 strain (11,930 nucleotides), amino acid in the Gprotein (333Aa) has been altered; the corresponding nucleic acids are atpositions 4370 to 4372.

SEQ ID NO: 9. ERA- (11,577 nucleotides), without the psi (pseudo-gene)region; an extra transcription unit has been introduced at nucleotidepositions 4950 to 5008.

SEQ ID NO: 10. ERA-2G (13,150 nucleotides), this strain has two copiesof the G gene; the second copy is inserted at positions 4988 to 6559.

SEQ ID NO: 11. ERAgreen (12,266 nucleotides), this strain contains thecoding sequence for GFP at positions 4993 to 5673; it appears greenunder UV light after infection of cells or tissue.

SEQ ID NO: 12. ERA-G (10,288 nucleotides), this strain has no G gene.

SEQ ID NO: 13. ERA-2g3 (13,150 nucleotides); this strain has two copiesof the G gene (the second of which is at positions 4988 to 6559), bothof which are substituted at amino acid 333 (corresponding to nucleotidepositions 4370-4372 and 6041-6043 in the shown sequence).

SEQ ID NO: 14. ERA-pt (11,976 nucleotides, with an extra transcriptionunit after the P gene, at positions 2469 to 2521).

SEQ ID NO: 15. ERA-pt-GFP (12,662 nucleotides, with GFP gene insertedafter P gene at 2505 to 3185).

SEQ ID NO: 16. ERAgm (11,914 nucleotides) positions of G and M genes areswitched with G at positions 2505-4076 and M at positions 4122-4727,respectively.

SEQ ID NO: 17. ERAg3m (11,914 nucleotides) positions of G and M genesare switched with G at positions 2505-4076 and M at positions 4122-4727,respectively. The G gene is mutated at amino acid position 333.

SEQ ID NO: 18. ERAgmg (13,556 nucleotides), this strain has two copiesof the G gene at positions 2505-4076 and 4943-6514, flanking the M geneat positions 4122-4727.

SEQ ID NO: 19. First ten nucleotides of hammerhead ribozymecorresponding to 5′ end of rabies virus ERA genome.

SEQ ID NO: 20. Nucleotide sequence encoding the SV40 T antigen nuclearlocalization signal (NLS).

SEQ ID NOs: 21-23. Artificial Kozak sequences.

SEQ ID NOs: 24-57. Synthetic oligonucleotides.

SEQ ID NO: 58. Amino acid sequence of G protein mutated at amino acidposition 333 (from Arg to Glu).

SEQ ID NOs: 59-65. Synthetic oligonucleotides.

DETAILED DESCRIPTION I. Introduction

Viral zoonoses are difficult to prevent. One major paradigm is thecontrol of wildlife rabies by oral vaccination. All current licensedoral rabies vaccines are based on one common source. The fixed rabiesvirus (RV) of Evelyn-Rokitnicki-Abelseth (ERA) was derived from theStreet-Alabama-Dufferin (SAD) strain, first isolated from a rabid dog inAlabama (USA) in 1935. The ERA strain was derived after multiplepassages of SAD RV in mouse brains, baby hamster kidney (BHK) cells, andchicken embryos. Repeated cloning of ERA in BHK cells resultedeventually in a B-19 clone, which was named SAD-B 19 for vaccinestudies. The first RV strain recovered by reverse genetics was SAD-B 19.Although SAD-B 19 and ERA RV derived from the same source, differentoutcomes have been observed in various animal oral vaccine studies. Forexample, ERA did not induce obvious neutralizing antibodies in eitherskunks or raccoons per os, while SAD-B 19 did. To elucidate potentialdifferences between these two RV strains, a reverse genetics system forthe ERA RV strain is required.

Reverse genetics presents a feasible way to modify RNA viruses indefined ways. A system for reverse genetics of an initial strain ofrabies virus was successfully established in 1994 (Schnell et al., TheEMBO J. 13, 4195-4203, 1994). In the intervening decade, improvements tothe system have been made, resulting in increased efficiency of virusrecovery. This increased efficiency has facilitated the elucidation ofvirus pathogenicity, protein-protein, and protein-RNA interactions.

Within the rabies virus genome, it has been proposed that some regionscontain important signals, such as viral distal promoters region,nucleoprotein encapsidation, RNA dependent RNA polymerase Ltranscription initiation site, polyadenylation and termination sites.These signals are important for ensuring efficient recovery of virus andfor designing an extra transcription unit for accepting an exogenousOpen Reading Frame (ORF) into the rabies virus genome.

This disclosure provides an efficient reverse genetics system, anddescribes its use to produce variants of the ERA strain virus.Modifications described herein have resulted in strains that aresuitable candidates for accepting ORF expression and vaccinedevelopment.

The reverse genetics system is composed of a set of plasmids. A firstplasmid includes an ERA viral cRNA. In order to create authentic viralanti-genomic ends in transcribed viral cDNA, ERA genomic cDNA is flankedby a hammerhead ribozyme at the 3′ end and a hepatitis delta virusribozyme at the 5′ end. The antigenomic cassette is fused to thebacteria phage T7 transcription initiation signal, which is optionallyalso under the control of cytomegalovirus (CMV) immediate-earlypromoter.

The system also includes a plurality of helper plasmids that encodeproteins involved in viral encapsidation. For example, the systemtypically includes helper plasmids that encode the viral nucleoprotein(N), phosphoprotein (P), RNA dependent polymerase (L), and optionallythe viral glycoprotein (G). The system also includes a plasmid thatencodes the phage T7 RNA polymerase (T7), which can be modified by theaddition of a nuclear localization signal (NLS) to increase expressionof the T7 polymerase in the nucleus of transfected cells. The T7 RNApolymerase expression plasmid is constructed as an “autogene,” whichtranscribes the whole length of viral anti-genomic cRNA fornucleoprotein encapsidation after transfection into cells.

The reverse genetics system is useful in the design and production ofimmunogenic compositions for the treatment (pre and/or post exposure) ofrabies virus, and for producing rabies virus ERA vectors for expressingexogenous Open Reading Frames (ORFs). For example, an extratranscription unit can be designed, tested and incorporated into the ERAgenome at either the Psi-region and/or at phosphoprotein (P)-matrix (M)protein intergenic region. Essentially any ORF of interest can beexpressed in the context to the ERA vector, including ORFs encodingantigens of viruses and other pathogens, such as antigens of otherlyssaviruses, as well as for expressing other proteins of therapeuticinterest.

Thus, the methods and compositions disclosed herein are useful for thedesign and production of rabies virus immunogenic compositions,including compositions suitable as vaccines for the pre and/or postexposure treatment of rabies virus.

II. Abbreviations

ADE antibody-dependant enhancement

Ag-ELISA antigen-capture ELISA

DNA deoxyribonucleic acid

ERA Rabies virus strain Evelyn-Rokitnicki-Abelseth

ELISA enzyme-linked immunosorbent assay

G glycoprotein

i.c. intracerebral

IFA indirect immunofluorescence assay

i.m. intramuscular

L RNA-dependent RNA-polymerase

M matrix protein

mAb monoclonal antibody

N nucleoprotein

ORF open reading frame

P phosphoprotein

PCR polymerase chain reaction

RACE 5′ rapid amplification of cDNA ends

RNA ribonucleic acid

RNP ribonucleoprotein

RT-PCR reverse transcription-polymerase chain reaction

RV rabies virus

trans 1 extra transcription unit 1

trans 2 extra transcription unit 2

III. Terms

Unless otherwise explained, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention belongs. Similarly, unlessotherwise noted, technical terms are used according to conventionalusage. Definitions of common terms in molecular biology may be found inBenjamin Lewin, Genes V, published by Oxford University Press, 1994(ISBN 0-19-854287-9); Kendrew et al. (eds.), The Encyclopedia ofMolecular Biology, published by Blackwell Science Ltd., 1994 (ISBN0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biology andBiotechnology: a Comprehensive Desk Reference, published by VCHPublishers, Inc., 1995 (ISBN 1-56081-569-8).

The singular terms “a,” “an,” and “the” include plural referents unlesscontext clearly indicates otherwise. Similarly, the word “or” isintended to include “and” unless the context clearly indicatesotherwise. Hence “comprising A or B” means including A, or B, or A andB. It is further to be understood that all base sizes or amino acidsizes, and all molecular weight or molecular mass values, given fornucleic acids or polypeptides are approximate, and are provided fordescription.

In order to facilitate review of the various embodiments of theinvention, the following explanations of specific terms are provided:

Adjuvant: A substance that non-specifically enhances the immune responseto an antigen. Development of vaccine adjuvants for use in humans isreviewed in Singh et al. (Nat. Biotechnol. 17:1075-1081, 1999), whichdiscloses that, at the time of its publication, aluminum salts and theMF59 microemulsion are the only vaccine adjuvants approved for humanuse.

Amplification: Amplification of a nucleic acid molecule (e.g., a DNA orRNA molecule) refers to use of a laboratory technique that increases thenumber of copies of a nucleic acid molecule in a sample. An example ofamplification is the polymerase chain reaction (PCR), in which a sampleis contacted with a pair of oligonucleotide primers under conditionsthat allow for the hybridization of the primers to a nucleic acidtemplate in the sample. The primers are extended under suitableconditions, dissociated from the template, re-annealed, extended, anddissociated to amplify the number of copies of the nucleic acid. Theproduct of amplification can be characterized by such techniques aselectrophoresis, restriction endonuclease cleavage patterns,oligonucleotide hybridization or ligation, and/or nucleic acidsequencing.

Other examples of amplification methods include strand displacementamplification, as disclosed in U.S. Pat. No. 5,744,311;transcription-free isothermal amplification, as disclosed in U.S. Pat.No. 6,033,881; repair chain reaction amplification, as disclosed in WO90/01069; ligase chain reaction amplification, as disclosed inEP-A-320,308; gap filling ligase chain reaction amplification, asdisclosed in U.S. Pat. No. 5,427,930; and NASBA™ RNA transcription-freeamplification, as disclosed in U.S. Pat. No. 6,025,134. An amplificationmethod can be modified, including for example by additional steps orcoupling the amplification with another protocol.

Animal: Living multi-cellular vertebrate organisms, a category thatincludes, for example, mammals and birds. The term mammal includes bothhuman and non-human mammals. Similarly, the term “subject” includes bothhuman and veterinary subjects, for example, humans, non-human primates,dogs, cats, horses, and cows.

Antibody: A protein (or protein complex) that includes one or morepolypeptides substantially encoded by immunoglobulin genes or fragmentsof immunoglobulin genes. The recognized immunoglobulin genes include thekappa, lambda, alpha, gamma, delta, epsilon, and mu constant regiongenes, as well as the myriad immunoglobulin variable region genes. Lightchains are classified as either kappa or lambda. Heavy chains areclassified as gamma, mu, alpha, delta, or epsilon, which in turn definethe immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.

The basic immunoglobulin (antibody) structural unit is generally atetramer. Each tetramer is composed of two identical pairs ofpolypeptide chains, each pair having one “light” (about 25 kDa) and one“heavy” (about 50-70 kDa) chain. The N-terminus of each chain defines avariable region of about 100 to 110 or more amino acids primarilyresponsible for antigen recognition. The terms “variable light chain”(V_(L)) and “variable heavy chain” (V_(H)) refer, respectively, to theselight and heavy chains.

As used herein, the term “antibody” includes intact immunoglobulins aswell as a number of well-characterized fragments. For instance, Fabs,Fvs, and single-chain Fvs (SCFvs) that bind to target protein (orepitope within a protein or fusion protein) would also be specificbinding agents for that protein (or epitope). These antibody fragmentsare as follows: (1) Fab, the fragment which contains a monovalentantigen-binding fragment of an antibody molecule produced by digestionof whole antibody with the enzyme papain to yield an intact light chainand a portion of one heavy chain; (2) Fab′, the fragment of an antibodymolecule obtained by treating whole antibody with pepsin, followed byreduction, to yield an intact light chain and a portion of the heavychain; two Fab′ fragments are obtained per antibody molecule; (3)(Fab′)₂, the fragment of the antibody obtained by treating wholeantibody with the enzyme pepsin without subsequent reduction; (4)F(ab′)₂, a dimer of two Fab′ fragments held together by two disulfidebonds; (5) Fv, a genetically engineered fragment containing the variableregion of the light chain and the variable region of the heavy chainexpressed as two chains; and (6) single chain antibody, a geneticallyengineered molecule containing the variable region of the light chain,the variable region of the heavy chain, linked by a suitable polypeptidelinker as a genetically fused single chain molecule. Methods of makingthese fragments are routine (see, for example, Harlow and Lane, UsingAntibodies: A Laboratory Manual, CSHL, New York, 1999).

Antibodies for use in the methods and compositions of this disclosurecan be monoclonal or polyclonal. Merely by way of example, monoclonalantibodies can be prepared from murine hybridomas according to theclassical method of Kohler and Milstein (Nature 256:495-97, 1975) orderivative methods thereof. Detailed procedures for monoclonal antibodyproduction are described in Harlow and Lane, Using Antibodies: ALaboratory Manual, CSHL, New York, 1999.

Antibody binding affinity: The strength of binding between a singleantibody binding site and a ligand (e.g., an antigen or epitope). Theaffinity of an antibody binding site X for a ligand Y is represented bythe dissociation constant (K_(d)), which is the concentration of Y thatis required to occupy half of the binding sites of X present in asolution. A smaller (K_(d)) indicates a stronger or higher-affinityinteraction between X and Y and a lower concentration of ligand isneeded to occupy the sites. In general, antibody binding affinity can beaffected by the alteration, modification and/or substitution of one ormore amino acids in the epitope recognized by the antibody paratope.

In one example, antibody binding affinity is measured by end-pointtitration in an Ag-ELISA assay. Antibody binding affinity issubstantially lowered (or measurably reduced) by the modification and/orsubstitution of one or more amino acids in the epitope recognized by theantibody paratope if the end-point titer of a specific antibody for themodified/substituted epitope differs by at least 4-fold, such as atleast 10-fold, at least 100-fold or greater, as compared to theunaltered epitope.

Antigen: A compound, composition, or substance that can stimulate theproduction of antibodies or a T-cell response in an animal, includingcompositions that are injected or absorbed into an animal. An antigenreacts with the products of specific humoral or cellular immunity,including those induced by heterologous immunogens. In one embodiment,an antigen is a virus antigen.

Attenuated: In the context of a live virus, such as a rabies virus, thevirus is attenuated if its ability to infect a cell or subject and/orits ability to produce disease is reduced (for example, eliminated).Typically, an attenuated virus retains at least some capacity to elicitan immune response following administration to an immunocompetentsubject. In some cases, an attenuated virus is capable of eliciting aprotective immune response without causing any signs or symptoms ofinfection.

Binding or Stable Binding: An oligonucleotide binds or stably binds to atarget nucleic acid if a sufficient amount of the oligonucleotide formsbase pairs or is hybridized to its target nucleic acid, to permitdetection of that binding. Binding can be detected by either physical orfunctional properties of the target:oligonucleotide complex. Bindingbetween a target and an oligonucleotide can be detected by any procedureknown to one skilled in the art, including functional or physicalbinding assays. Binding can be detected functionally by determiningwhether binding has an observable effect upon a biosynthetic processsuch as expression of a gene, DNA replication, transcription,translation, and the like.

Physical methods of detecting the binding of complementary strands ofDNA or RNA are well known in the art, and include such methods as DNaseI or chemical footprinting, gel shift and affinity cleavage assays,Northern blotting, Southern blotting, dot blotting, and light absorptiondetection procedures. For example, a method which is widely used,because it is so simple and reliable, involves observing a change inlight absorption of a solution containing an oligonucleotide (or ananalog) and a target nucleic acid at 220 to 300 nm as the temperature isslowly increased. If the oligonucleotide or analog has bound to itstarget, there is a sudden increase in absorption at a characteristictemperature as the oligonucleotide (or analog) and target dissociate ormelt.

The binding between an oligomer and its target nucleic acid isfrequently characterized by the temperature (T_(m)) at which 50% of theoligomer is melted from its target. A higher T_(m) means a stronger ormore stable complex relative to a complex with a lower T_(m).

cDNA (complementary DNA): A piece of DNA lacking internal, non-codingsegments (introns) and regulatory sequences that determinetranscription. cDNA is synthesized in the laboratory by reversetranscription from messenger RNA extracted from cells.

Electrophoresis: Electrophoresis refers to the migration of chargedsolutes or particles in a liquid medium under the influence of anelectric field. Electrophoretic separations are widely used for analysisof macromolecules. Of particular importance is the identification ofproteins and nucleic acid sequences. Such separations can be based ondifferences in size and/or charge. Nucleotide sequences have a uniformcharge and are therefore separated based on differences in size.Electrophoresis can be performed in an unsupported liquid medium (forexample, capillary electrophoresis), but more commonly the liquid mediumtravels through a solid supporting medium. The most widely usedsupporting media are gels, for example, polyacrylamide and agarose gels.

Sieving gels (for example, agarose) impede the flow of molecules. Thepore size of the gel determines the size of a molecule that can flowfreely through the gel. The amount of time to travel through the gelincreases as the size of the molecule increases. As a result, smallmolecules travel through the gel more quickly than large molecules andthus progress further from the sample application area than largermolecules, in a given time period. Such gels are used for size-basedseparations of nucleotide sequences.

Fragments of linear DNA migrate through agarose gels with a mobilitythat is inversely proportional to the log₁₀ of their molecular weight.By using gels with different concentrations of agarose, different sizesof DNA fragments can be resolved. Higher concentrations of agarosefacilitate separation of small DNAs, while low agarose concentrationsallow resolution of larger DNAs.

Epitope: An antigenic determinant. These are particular chemical groups,such as contiguous or non-contiguous peptide sequences, on a moleculethat are antigenic, that is, that elicit a specific immune response. Anantibody binds a particular antigenic epitope based on the threedimensional structure of the antibody and the matching (or cognate)three dimensional structure of the epitope.

A “substituted epitope” comprises at least one structural substitutionin the epitope, such as a substitution of one amino acid for another

Hybridization: Oligonucleotides and their analogs hybridize by hydrogenbonding, which includes Watson-Crick, Hoogsteen or reversed Hoogsteenhydrogen bonding, between complementary bases. Generally, nucleic acidconsists of nitrogenous bases that are either pyrimidines (cytosine (C),uracil (U), and thymine (T)) or purines (adenine (A) and guanine (G)).These nitrogenous bases form hydrogen bonds between a pyrimidine and apurine, and the bonding of the pyrimidine to the purine is referred toas “base pairing.” More specifically, A will hydrogen bond to T or U,and G will bond to C. “Complementary” refers to the base pairing thatoccurs between to distinct nucleic acid sequences or two distinctregions of the same nucleic acid sequence.

“Specifically hybridizable” and “specifically complementary” are termsthat indicate a sufficient degree of complementarity such that stableand specific binding occurs between the oligonucleotide (or its analog)and the DNA or RNA target. The oligonucleotide or oligonucleotide analogneed not be 100% complementary to its target sequence to be specificallyhybridizable. An oligonucleotide or analog is specifically hybridizablewhen binding of the oligonucleotide or analog to the target DNA or RNAmolecule interferes with the normal function of the target DNA or RNA,and there is a sufficient degree of complementarity to avoidnon-specific binding of the oligonucleotide or analog to non-targetsequences under conditions where specific binding is desired, forexample under physiological conditions in the case of in vivo assays orsystems. Such binding is referred to as specific hybridization.

Hybridization conditions resulting in particular degrees of stringencywill vary depending upon the nature of the hybridization method ofchoice and the composition and length of the hybridizing nucleic acidsequences. Generally, the temperature of hybridization and the ionicstrength (especially the Na⁺ and/or Mg⁺⁺ concentration) of thehybridization buffer will determine the stringency of hybridization,though wash times also influence stringency. Calculations regardinghybridization conditions required for attaining particular degrees ofstringency are discussed by Sambrook et al. (ed.), Molecular Cloning: ALaboratory Manual, 2^(nd) ed., vol. 1-3, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1989, chapters 9 and 11; and Ausubel etal. Short Protocols in Molecular Biology, 4^(th) ed., John Wiley & Sons,Inc., 1999.

For purposes of the present disclosure, “stringent conditions” encompassconditions under which hybridization will only occur if there is lessthan 25% mismatch between the hybridization molecule and the targetsequence. “Stringent conditions” may be broken down into particularlevels of stringency for more precise definition. Thus, as used herein,“moderate stringency” conditions are those under which molecules withmore than 25% sequence mismatch will not hybridize; conditions of“medium stringency” are those under which molecules with more than 15%mismatch will not hybridize, and conditions of “high stringency” arethose under which sequences with more than 10% mismatch will nothybridize. Conditions of “very high stringency” are those under whichsequences with more than 6% mismatch will not hybridize.

“Specific hybridization” refers to the binding, duplexing, orhybridizing of a molecule only or substantially only to a particularnucleotide sequence when that sequence is present in a complex mixture(for example, total cellular DNA or RNA). Specific hybridization mayalso occur under conditions of varying stringency.

Immune stimulatory composition: A term used herein to mean a compositionuseful for stimulating or eliciting a specific immune response (orimmunogenic response) in a vertebrate. The immune stimulatorycomposition can be a protein antigen or a plasmid vector used to expressa protein antigen. In some embodiments, the immunogenic response isprotective or provides protective immunity, in that it enables thevertebrate animal to better resist infection with or disease progressionfrom the organism against which the immune stimulatory composition isdirected.

Without wishing to be bound by a specific theory, it is believed that animmunogenic response induced by an immune stimulatory composition mayarise from the generation of an antibody specific to one or more of theepitopes provided in the immune stimulatory composition. Alternatively,the response may comprise a T-helper or cytotoxic cell-based response toone or more of the epitopes provided in the immune stimulatorycomposition. All three of these responses may originate from naïve ormemory cells. One specific example of a type of immune stimulatorycomposition is a vaccine.

In some embodiments, an “effective amount” or “immune-stimulatoryamount” of an immune stimulatory composition is an amount which, whenadministered to a subject, is sufficient to engender a detectable immuneresponse. Such a response may comprise, for instance, generation of anantibody specific to one or more of the epitopes provided in the immunestimulatory composition. Alternatively, the response may comprise aT-helper or CTL-based response to one or more of the epitopes providedin the immune stimulatory composition. All three of these responses mayoriginate from naïve or memory cells. In other embodiments, a“protective effective amount” of an immune stimulatory composition is anamount which, when administered to a subject, is sufficient to conferprotective immunity upon the subject.

Inhibiting or treating a disease: Inhibiting the full development of adisease or condition, for example, in a subject who is at risk for adisease. A specific example of diseases is rabies. “Treatment” refers toa therapeutic intervention that ameliorates a sign or symptom of adisease or pathological condition after it has begun to develop. As usedherein, the term “ameliorating,” with reference to a disease,pathological condition or symptom, refers to any observable beneficialeffect of the treatment. The beneficial effect can be evidenced, forexample, by a delayed onset of clinical symptoms of the disease in asusceptible subject, a reduction in severity of some or all clinicalsymptoms of the disease, a slower progression of the disease, areduction in the number of relapses of the disease, an improvement inthe overall health or well-being of the subject, or by other parameterswell known in the art that are specific to the particular disease.

Isolated: An “isolated” or “purified” biological component (such as anucleic acid, peptide, protein, protein complex, or particle) has beensubstantially separated, produced apart from, or purified away fromother biological components in the cell of the organism in which thecomponent naturally occurs, that is, other chromosomal andextra-chromosomal DNA and RNA, and proteins. Nucleic acids, peptides andproteins that have been “isolated” or “purified” thus include nucleicacids and proteins purified by standard purification methods. The termalso embraces nucleic acids, peptides and proteins prepared byrecombinant expression in a host cell, as well as chemically synthesizednucleic acids or proteins. The term “isolated” or “purified” does notrequire absolute purity; rather, it is intended as a relative term.Thus, for example, an isolated biological component is one in which thebiological component is more enriched than the biological component isin its natural environment within a cell, or other production vessel.Preferably, a preparation is purified such that the biological componentrepresents at least 50%, such as at least 70%, at least 90%, at least95%, or greater, of the total biological component content of thepreparation.

Label: A detectable compound or composition that is conjugated directlyor indirectly to another molecule to facilitate detection of thatmolecule. Specific, non-limiting examples of labels include fluorescenttags, enzymatic linkages, and radioactive isotopes.

Nucleic acid molecule: A polymeric form of nucleotides, which mayinclude both sense and anti-sense strands of RNA, cDNA, genomic DNA, andsynthetic forms and mixed polymers of the above. A nucleotide refers toa ribonucleotide, deoxynucleotide or a modified form of either type ofnucleotide. The term “nucleic acid molecule” as used herein issynonymous with “nucleic acid” and “polynucleotide.” A nucleic acidmolecule is usually at least 10 bases in length, unless otherwisespecified. The term includes single- and double-stranded forms of DNA. Apolynucleotide may include either or both naturally occurring andmodified nucleotides linked together by naturally occurring and/ornon-naturally occurring nucleotide linkages.

Oligonucleotide: A nucleic acid molecule generally comprising a lengthof 300 bases or fewer. The term often refers to single-strandeddeoxyribonucleotides, but it can refer as well to single- ordouble-stranded ribonucleotides, RNA:DNA hybrids and double-strandedDNAs, among others. The term “oligonucleotide” also includesoligonucleosides (that is, an oligonucleotide minus the phosphate) andany other organic base polymer.

In some examples, oligonucleotides are about 10 to about 90 bases inlength, for example, 12, 13, 14, 15, 16, 17, 18, 19 or 20 bases inlength. Other oligonucleotides are about 25, about 30, about 35, about40, about 45, about 50, about 55, about 60 bases, about 65 bases, about70 bases, about 75 bases or about 80 bases in length. Oligonucleotidesmay be single-stranded, for example, for use as probes or primers, ormay be double-stranded, for example, for use in the construction of amutant gene. Oligonucleotides can be either sense or anti-senseoligonucleotides. An oligonucleotide can be modified as discussed abovein reference to nucleic acid molecules. Oligonucleotides can be obtainedfrom existing nucleic acid sources (for example, genomic or cDNA), butcan also be synthetic (for example, produced by laboratory or in vitrooligonucleotide synthesis).

Open Reading Frame (ORF): A series of nucleotide triplets (codons)coding for amino acids without any internal termination codons. Thesesequences are usually translatable into apeptide/polypeptide/protein/polyprotein.

It is recognized in the art that the following codons (shown for RNA)can be used interchangeably to code for each specific amino acid ortermination: Alanine (Ala or A) GCU, GCG, GCA, or GCG; Arginine (Arg orR) CGU, CGC, CGA, CGG, AGA, or AGG; Asparagine (Asn or N) AAU or AAC;Aspartic Acid (Asp or D) GAU or GAC; Cysteine (Cys or C) UGU or UGC;Glutamic Acid (Glu or E) GAA or GAG; Glutamine (Gln or Q) CAA or CAG;Glycine (Gly or G) GGU, GGC, GGA, or GGG; Histidine (His or H) CAU orCAC; Isoleucine (Ile or I) AUU, AUC, or AUA; Leucine (Leu or L) UUA,UUG, CUU, CUC, CUA, or CUG; Lysine (Lys or K) AAA or AAG; Methionine(Met or M) AUG; Phenylalanine (Phe or F) UUU or UUC; Proline (Pro or P)CCU, CCC, CCA, or CCG; Serine (Ser or S) UCU, UCC, UCA, UCG, AGU, orAGC; Termination codon UAA (ochre) or UAG (amber) or UGA (opal);Threonine (Thr or T) ACU, ACC, ACA, or ACG; Tyrosine (Tyr or Y) UAU orUAC; Tryptophan (Trp or W) UGG; and Valine (Val or V) GUU, GUC, GUA, orGUG. The corresponding codons for DNA have T substituted for U in eachinstance.

Operably linked: A first nucleic acid sequence is operably linked with asecond nucleic acid sequence when the first nucleic acid sequence isplaced in a functional relationship with the second nucleic acidsequence. For instance, a promoter is operably linked to a codingsequence is the promoter affects the transcription or expression of thecoding sequence. Generally, operably linked DNA sequences are contiguousand, where necessary to join two protein coding regions, in the samereading frame. If introns are present, the operably linked DNA sequencesmay not be contiguous.

Paratope: That portion of an antibody that is responsible for itsbinding to an antigenic determinant (epitope) on an antigen.

Pharmaceutically Acceptable Carriers: The pharmaceutically acceptablecarriers useful in this disclosure are conventional. Remington'sPharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton,Pa., 15th Edition (1975), describes compositions and formulationssuitable for pharmaceutical delivery of one or more therapeuticcompounds or molecules, such as one or more SARS-CoV nucleic acidmolecules, proteins or antibodies that bind these proteins, andadditional pharmaceutical agents.

In general, the nature of the carrier will depend on the particular modeof administration being employed. For instance, parenteral formulationsusually comprise injectable fluids that include pharmaceutically andphysiologically acceptable fluids such as water, physiological saline,balanced salt solutions, aqueous dextrose, glycerol or the like as avehicle. For solid compositions (for example, powder, pill, tablet, orcapsule forms), conventional non-toxic solid carriers can include, forexample, pharmaceutical grades of mannitol, lactose, starch, ormagnesium stearate. In addition to biologically-neutral carriers,pharmaceutical compositions to be administered can contain minor amountsof non-toxic auxiliary substances, such as wetting or emulsifyingagents, preservatives, and pH buffering agents and the like, for examplesodium acetate or sorbitan monolaurate.

Polypeptide: A polymer in which the monomers are amino acid residuesjoined together through amide bonds. When the amino acids arealpha-amino acids, either the L-optical isomer or the D-optical isomercan be used, the L-isomers being preferred for many biological uses. Theterms “polypeptide” or “protein” as used herein are intended toencompass any amino acid molecule and include modified amino acidmolecules. The term “polypeptide” is specifically intended to covernaturally occurring proteins, as well as those which are recombinantlyor synthetically produced.

Conservative amino acid substitutions are those substitutions that, whenmade, least interfere with the properties of the original protein, thatis, the structure and especially the function of the protein isconserved and not significantly changed by such substitutions. Examplesof conservative substitutions are shown below.

Original Residue Conservative Substitutions Ala Ser Arg Lys Asn Gln, HisAsp Glu Cys Ser Gln Asn Glu Asp His Asn; Gln Ile Leu, Val Leu Ile; ValLys Arg; Gln; Glu Met Leu; Ile Phe Met; Leu; Tyr Ser Thr Thr Ser Trp TyrTyr Trp; Phe Val Ile; Leu

Conservative substitutions generally maintain (a) the structure of thepolypeptide backbone in the area of the substitution, for example, as asheet or helical conformation, (b) the charge or hydrophobicity of themolecule at the target site, or (c) the bulk of the side chain.

Amino acids are typically classified in one or more categories,including polar, hydrophobic, acidic, basic and aromatic, according totheir side chains. Examples of polar amino acids include those havingside chain functional groups such as hydroxyl, sulfhydryl, and amide, aswell as the acidic and basic amino acids. Polar amino acids include,without limitation, asparagine, cysteine, glutamine, histidine,selenocysteine, serine, threonine, tryptophan and tyrosine. Examples ofhydrophobic or non-polar amino acids include those residues havingnonpolar aliphatic side chains, such as, without limitation, leucine,isoleucine, valine, glycine, alanine, proline, methionine andphenylalanine. Examples of basic amino acid residues include thosehaving a basic side chain, such as an amino or guanidino group. Basicamino acid residues include, without limitation, arginine, homolysineand lysine. Examples of acidic amino acid residues include those havingan acidic side chain functional group, such as a carboxy group. Acidicamino acid residues include, without limitation aspartic acid andglutamic acid. Aromatic amino acids include those having an aromaticside chain group. Examples of aromatic amino acids include, withoutlimitation, biphenylalanine, histidine, 2-napthylalananine,pentafluorophenylalanine, phenylalanine, tryptophan and tyrosine. It isnoted that some amino acids are classified in more than one group, forexample, histidine, tryptophan, and tyrosine are classified as bothpolar and aromatic amino acids. Additional amino acids that areclassified in each of the above groups are known to those of ordinaryskill in the art.

Substitutions which in general are expected to produce the greatestchanges in protein properties will be non-conservative, for instancechanges in which (a) a hydrophilic residue, for example, seryl orthreonyl, is substituted for (or by) a hydrophobic residue, for example,leucyl, isoleucyl, phenylalanyl, valyl or alanyl; (b) a cysteine orproline is substituted for (or by) any other residue; (c) a residuehaving an electropositive side chain, for example, lysyl, arginyl, orhistadyl, is substituted for (or by) an electronegative residue, forexample, glutamyl or aspartyl; or (d) a residue having a bulky sidechain, for example, phenylalanine, is substituted for (or by) one nothaving a side chain, for example, glycine.

Probes and primers: A probe comprises an isolated nucleic acid moleculeattached to a detectable label or other reporter molecule. Typicallabels include radioactive isotopes, enzyme substrates, co-factors,ligands, chemiluminescent or fluorescent agents, haptens, and enzymes.Methods for labeling and guidance in the choice of labels appropriatefor various purposes are discussed, for example, in Sambrook et al.(ed.), Molecular Cloning: A Laboratory Manual, 2^(nd) ed., vol. 1-3,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989 andAusubel et al. Short Protocols in Molecular Biology, 4^(th) ed., JohnWiley & Sons, Inc., 1999.

Primers are short nucleic acid molecules, for instance DNAoligonucleotides 6 nucleotides or more in length, for example thathybridize to contiguous complementary nucleotides or a sequence to beamplified. Longer DNA oligonucleotides may be about 10, 12, 15, 20, 25,30, or 50 nucleotides or more in length. Primers can be annealed to acomplementary target DNA strand by nucleic acid hybridization to form ahybrid between the primer and the target DNA strand, and then the primerextended along the target DNA strand by a DNA polymerase enzyme. Primerpairs can be used for amplification of a nucleic acid sequence, forexample, by the polymerase chain reaction (PCR) or other nucleic-acidamplification methods known in the art. Other examples of amplificationinclude strand displacement amplification, as disclosed in U.S. Pat. No.5,744,311; transcription-free isothermal amplification, as disclosed inU.S. Pat. No. 6,033,881; repair chain reaction amplification, asdisclosed in WO 90/01069; ligase chain reaction amplification, asdisclosed in EP-A-320 308; gap filling ligase chain reactionamplification, as disclosed in U.S. Pat. No. 5,427,930; and NASBA™ RNAtranscription-free amplification, as disclosed in U.S. Pat. No.6,025,134.

Methods for preparing and using nucleic acid probes and primers aredescribed, for example, in Sambrook et al. (ed.), Molecular Cloning: ALaboratory Manual, 2^(nd) ed., vol. 1-3, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1989; Ausubel et al. Short Protocols inMolecular Biology, 4^(th) ed., John Wiley & Sons, Inc., 1999; and Inniset al. PCR Protocols, A Guide to Methods and Applications, AcademicPress, Inc., San Diego, Calif., 1990. Amplification primer pairs can bederived from a known sequence, for example, by using computer programsintended for that purpose such as Primer (Version 0.5, © 1991, WhiteheadInstitute for Biomedical Research, Cambridge, Mass.). One of ordinaryskill in the art will appreciate that the specificity of a particularprobe or primer increases with its length. Thus, in order to obtaingreater specificity, probes and primers can be selected that comprise atleast 20, 25, 30, 35, 40, 45, 50 or more consecutive nucleotides of atarget nucleotide sequences.

Protein: A biological molecule, particularly a polypeptide, expressed bya gene and comprised of amino acids.

Purified: The term “purified” does not require absolute purity; rather,it is intended as a relative term. Thus, for example, a purified proteinpreparation is one in which the subject protein is more pure than in itsnatural environment within a cell. Generally, a protein preparation ispurified such that the protein represents at least 50% of the totalprotein content of the preparation.

Recombinant nucleic acid: A nucleic acid molecule that is not naturallyoccurring or has a sequence that is made by an artificial combination oftwo otherwise separated segments of sequence. This artificialcombination is accomplished by chemical synthesis or, more commonly, bythe artificial manipulation of isolated segments of nucleic acids, e.g.,by genetic engineering techniques such as those described in Sambrook etal. (ed.), Molecular Cloning: A Laboratory Manual, 2^(nd) ed., vol. 1-3,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989. Theterm recombinant includes nucleic acids that have been altered solely byaddition, substitution, or deletion of a portion of a natural nucleicacid molecule.

Regulatory sequences or elements: These terms refer generally to a classof DNA sequences that influence or control expression of genes. Includedin the term are promoters, enhancers, locus control regions (LCRs),insulators/boundary elements, silencers, matrix attachment regions(MARs, also referred to as scaffold attachment regions), repressor,transcriptional terminators, origins of replication, centromeres, andmeiotic recombination hotspots. Promoters are sequences of DNA near the5′-end of a gene that act as a binding site for DNA-dependent RNApolymerase, and from which transcription is initiated. Enhancers arecontrol elements that elevate the level of transcription from apromoter, usually independently of the enhancer's orientation ordistance from the promoter. LCRs confer tissue-specific and temporallyregulated expression to genes to which they are linked. LCRs functionindependently of their position in relation to the gene, but arecopy-number dependent. It is believed that they function to open thenucleosome structure, so other factors can bind to the DNA. LCRs mayalso affect replication timing and origin usage. Insulators (also knowas boundary elements) are DNA sequences that prevent the activation (orinactivation) of transcription of a gene, by blocking effects ofsurrounding chromatin. Silencers and repressors are control elementsthat suppress gene expression; they act on a gene independently of theirorientation or distance from the gene. MARs are sequences within DNAthat bind to the nuclear scaffold; they can affect transcription,possibly by separating chromosomes into regulatory domains. It isbelieved that MARs mediate higher-order, looped structures withinchromosomes. Transcriptional terminators are regions within the genevicinity where RNA polymerase is released from the template. Origins ofreplication are regions of the genome that, during DNA synthesis orreplication phases of cell division, begin the replication process ofDNA. Meiotic recombination hotspots are regions of the genome thatrecombine more frequently than average during meiosis.

Replicon: Any genetic element (e.g., plasmid, chromosome, virus) thatfunctions as an autonomous, self-replicating unit of DNA replication invivo.

Sample: A portion, piece, or segment that is representative of a whole.This term encompasses any material, including for instance samplesobtained from an animal, a plant, or the environment.

An “environmental sample” includes a sample obtained from inanimateobjects or reservoirs within an indoor or outdoor environment.Environmental samples include, but are not limited to: soil, water,dust, and air samples; bulk samples, including building materials,furniture, and landfill contents; and other reservoir samples, such asanimal refuse, harvested grains, and foodstuffs.

A “biological sample” is a sample obtained from a plant or animalsubject. As used herein, biological samples include all samples usefulfor detection of viral infection in subjects, including, but not limitedto: cells, tissues, and bodily fluids, such as blood; derivatives andfractions of blood (such as serum); extracted galls; biopsied orsurgically removed tissue, including tissues that are, for example,unfixed, frozen, fixed in formalin and/or embedded in paraffin; tears;milk; skin scrapes; surface washings; urine; sputum; cerebrospinalfluid; prostate fluid; pus; bone marrow aspirates; BAL; saliva; cervicalswabs; vaginal swabs; and oropharyngeal wash.

Sequence identity: The similarity between two nucleic acid sequences, ortwo amino acid sequences, is expressed in terms of the similaritybetween the sequences, otherwise referred to as sequence identity.Sequence identity is frequently measured in terms of percentage identity(or similarity or homology); the higher the percentage, the more similarthe two sequences are.

Methods of alignment of sequences for comparison are well known in theart. Various programs and alignment algorithms are described in: Smithand Waterman (Adv. Appl. Math., 2:482, 1981); Needleman and Wunsch (J.Mol. Biol., 48:443, 1970); Pearson and Lipman (Proc. Natl. Acad. Sci.,85:2444, 1988); Higgins and Sharp (Gene, 73:237-44, 1988); Higgins andSharp (CABIOS, 5:151-53, 1989); Corpet et al. (Nuc. Acids Res.,16:10881-90, 1988); Huang et al. (Comp. Appls. Biosci., 8:155-65, 1992);and Pearson et al. (Meth. Mol. Biol., 24:307-31, 1994). Altschul et al.(Nature Genet., 6:119-29, 1994) presents a detailed consideration ofsequence alignment methods and homology calculations.

The alignment tools ALIGN (Myers and Miller, CABIOS 4:11-17, 1989) orLFASTA (Pearson and Lipman, 1988) may be used to perform sequencecomparisons (Internet Program © 1996, W. R. Pearson and the Universityof Virginia, “fasta20u63” version 2.0u63, release date December 1996).ALIGN compares entire sequences against one another, while LFASTAcompares regions of local similarity. These alignment tools and theirrespective tutorials are available on the Internet at the NCSA website.Alternatively, for comparisons of amino acid sequences of greater thanabout 30 amino acids, the “Blast 2 sequences” function can be employedusing the default BLOSUM62 matrix set to default parameters, (gapexistence cost of 11, and a per residue gap cost of 1). When aligningshort peptides (fewer than around 30 amino acids), the alignment shouldbe performed using the “Blast 2 sequences” function, employing the PAM30matrix set to default parameters (open gap 9, extension gap 1penalties). The BLAST sequence comparison system is available, forinstance, from the NCBI web site; see also Altschul et al., J. Mol.Biol., 215:403-10, 1990; Gish and States, Nature Genet., 3:266-72, 1993;Madden et al., Meth. Enzymol., 266:131-41, 1996; Altschul et al.,Nucleic Acids Res., 25:3389-402, 1997; and Zhang and Madden, GenomeRes., 7:649-56, 1997.

Orthologs (equivalent to proteins of other species) of proteins are insome instances characterized by possession of greater than 75% sequenceidentity counted over the full-length alignment with the amino acidsequence of specific protein using ALIGN set to default parameters.Proteins with even greater similarity to a reference sequence will showincreasing percentage identities when assessed by this method, such asat least 80%, at least 85%, at least 90%, at least 92%, at least 95%, orat least 98% sequence identity. In addition, sequence identity can becompared over the full length of one or both binding domains of thedisclosed fusion proteins.

When significantly less than the entire sequence is being compared forsequence identity, homologous sequences will typically possess at least80% sequence identity over short windows of 10-20, and may possesssequence identities of at least 85%, at least 90%, at least 95%, or atleast 99% depending on their similarity to the reference sequence.Sequence identity over such short windows can be determined usingLFASTA; methods are described at the NCSA website. One of skill in theart will appreciate that these sequence identity ranges are provided forguidance only; it is entirely possible that strongly significanthomologs could be obtained that fall outside of the ranges provided.Similar homology concepts apply for nucleic acids as are described forprotein. An alternative indication that two nucleic acid molecules areclosely related is that the two molecules hybridize to each other understringent conditions.

Nucleic acid sequences that do not show a high degree of identity maynevertheless encode similar amino acid sequences, due to the degeneracyof the genetic code. It is understood that changes in nucleic acidsequence can be made using this degeneracy to produce multiple nucleicacid sequences that each encode substantially the same protein.

Specific binding agent: An agent that binds substantially only to adefined target. Thus a protein-specific binding agent bindssubstantially only the defined protein, or to a specific region withinthe protein. As used herein, protein-specific binding agents includeantibodies and other agents that bind substantially to a specifiedpolypeptide. The antibodies may be monoclonal or polyclonal antibodiesthat are specific for the polypeptide, as well as immunologicallyeffective portions (“fragments”) thereof.

The determination that a particular agent binds substantially only to aspecific polypeptide may readily be made by using or adapting routineprocedures. Examples of suitable in vitro assays which make use of theWestern blotting procedure include IFA and Ag-ELISA, and are describedin many standard texts, including Harlow and Lane, Using Antibodies: ALaboratory Manual, CSHL, New York, 1999.

Transformed: A “transformed” cell is a cell into which has beenintroduced a nucleic acid molecule by molecular biology techniques. Theterm encompasses all techniques by which a nucleic acid molecule mightbe introduced into such a cell, including transfection with viralvectors, transformation with plasmid vectors, and introduction of nakedDNA by electroporation, lipofection, and particle gun acceleration.

Vector: A nucleic acid molecule as introduced into a host cell, therebyproducing a transformed host cell. A vector may include nucleic acidsequences that permit it to replicate in a host cell, such as an originof replication (DNA sequences that participate in initiating DNAsynthesis). A vector may also include one or more selectable markergenes and other genetic elements known in the art.

Virus: Microscopic infectious organism that reproduces inside livingcells. A virus typically consists essentially of a core of a singlenucleic acid surrounded by a protein coat, and has the ability toreplicate only inside a living cell. “Viral replication” is theproduction of additional virus by the occurrence of at least one virallife cycle. A virus may subvert the host cells' normal functions,causing the cell to behave in a manner determined by the virus. Forexample, a viral infection may result in a cell producing a cytokine, orresponding to a cytokine, when the uninfected cell does not normally doso.

Although methods and materials similar or equivalent to those describedherein can be used in the practice or testing of the present invention,suitable methods and materials are described below. All publications,patent applications, patents, and other references mentioned herein areincorporated by reference in their entirety. In case of conflict, thepresent specification, including explanations of terms, will control. Inaddition, the materials, methods, and examples are illustrative only andnot intended to be limiting.

IV. Overview of Several Embodiments

Provided herein in a first embodiment is a recombinant rabies virusgenome comprising the nucleic acid as set forth in SEQ ID NO: 1 (fulllength ERA sequence). Also provided are isolated rabies virus proteinsencoded by that genome, including specific proteins comprising an aminoacid sequence as set forth in SEQ ID NO: 2 (N protein); SEQ ID NO: 3 (Pprotein); SEQ ID NO: 4 (M protein); SEQ ID NO: 5 (G protein); or SEQ IDNO: 6 (L protein); and isolated nucleic acid molecules that encode suchproteins. By way of example, such isolated nucleic acid moleculescomprise a nucleotide sequence as set forth in: nucleotides 71-1423 ofSEQ ID NO: 1 (N protein); nucleotides 1511-2407 of SEQ ID NO: 1 (Pprotein); nucleotides 2491-3104 of SEQ ID NO: 1 (M protein); nucleotides3318-4892 of SEQ ID NO: 1 (G protein); or nucleotides 5418-11,801 of SEQID NO: 1 (L protein).

Also provided is a recombinant virus genome with the nucleic acidsequence as set forth in SEQ ID NO: 7, which differs from SEQ ID NO: 1by virtue of a deletion of one adenosine residue in the polyA tractbetween the G gene and the psi-region. SEQ ID NO: 7 also encodes theproteins shown as SEQ ID NOs: 2-6.

Also provided are genomes of derivatives of the ERA strain of virus, asshown in SEQ ID NOs: 8-18. In certain embodiments, the genomes arepresent in a vector, such as a plasmid. Yet another described embodimentis a system for sequencing full length rabies virus genome using amethod as described herein. Viral vector systems for expression ofheterologous proteins are also described.

Another embodiment provides compositions that comprise one or morenucleic acid molecules, or one or more proteins, provided herein.Optionally, such compositions contain a pharmaceutically acceptablecarrier, an adjuvant, or a combination of two or more thereof.

Also provided is a method of eliciting an immune response against anantigenic epitope in a subject, comprising introducing into the subjecta composition comprising a nucleotide, peptide, or polypeptide describedherein, thereby eliciting an immune response in the subject.

Another aspect of the disclosure relates to a vector system forproducing recombinant rabies virus. The vector system includes a firstvector (transcription vector) containing a full-length rabies virusantigenomic DNA (or a derivative thereof) and a set of helper vectorscontaining nucleic acids that encode at least one rabies virus strainERA protein. Expression of the vectors in a transfected host cellresults in production of a live recombinant rabies virus. In certainembodiments, the antigenomic DNA is of the ERA strain (for example, SEQID NO: 1 or SEQ ID NO: 7) or a derivative thereof, such as one of SEQ IDNOs: 8-18. In certain embodiments, the vectors are plasmids.

To facilitate recovery of full length viral RNA, the transcriptionvector can include, in a 5′ to 3′ direction: a hammerhead ribozyme; arabies virus antigenomic cDNA; and a hepatitis delta virus ribozyme.Nucleotides of the hammerhead ribozyme are selected to be complementaryto the antisense genomic sequence of the rabies virus. Transcription ofthe antigenomic cDNA is under the transcription regulatory control of atleast one of the CMV promoter and the phage T7 RNA polymerase promoter,and commonly under the control of both of these promoters.

The helper vectors typically include a vector comprising apolynucleotide sequence that encodes a rabies virus N protein; a vectorcomprising a polynucleotide sequence that encodes a rabies virus Pprotein; a vector comprising a polynucleotide sequence that encodes arabies virus M protein; a vector comprising a polynucleotide sequencethat encodes a rabies virus L protein; and a vector comprising apolynucleotide sequence that encodes a phage T7 RNA polymerase. In anembodiment, the T7 RNA polymerase comprises a nuclear localizationsignal (NLS). Optionally, the vector system also includes a vectorcomprising a polynucleotide sequence that encodes a rabies virus Gprotein.

Transcription of one or more of the polynucleotide sequences that encodethe rabies virus P, M, L or G protein or the T7 polymerase is under thetranscription regulatory control of both the CMV promoter and the T7promoter. In contrast, transcription of the polynucleotide sequence thatencodes the rabies virus N protein is under the transcription regulatorycontrol of the T7 promoter, and transcription is cap-independent.

Yet more embodiments are live rabies vaccines, each comprising arecombinant rabies virus genome as provided herein. Examples of suchrecombinant rabies genomes comprise the sequence shown as ERA G333 (SEQID NO: 13); the sequence shown as ERA 2G (SEQ ID NO: 8); and thesequence shown herein as ERA 2G333 (SEQ ID NO: 10). Optionally, therabies vaccine is attenuated.

Also provided is a method of producing a live rabies virus (for example,for use in an immunogenic composition, such as a vaccine) by introducingthe vector system into a host cell. After transfection of vector systeminto a suitable host cell, live and optionally attenuated virus isrecovered. Production and administration of a live rabies vaccineproduced by such methods is also contemplated herein.

Also disclosed is a method of vaccinating a subject against rabies,which method comprises administering an effective amount of the liverabies vaccine according to the provided description to a subject, suchthat cells of the subject are infected with the rabies vaccine, whereinan anti-rabies immune response is produced in the subject. In oneembodiment, the subject is a human. In another embodiment, the subjectis a non-human animal. For instance, the non-human animal in someinstances is a cat, dog, rat, mouse, bat, fox, raccoon, squirrel,opossum, coyote, or wolf.

In certain embodiments, the rabies vaccine is administered enterally.For instance, the enteral administration in some cases comprises oraladministration. Oral administration includes administration throughfood-baits designed to vaccinate wild animal populations, for instance.

Pharmaceutical compositions that include the described live rabiesvaccines (for instance, an attenuated live rabies vaccine) and apharmaceutically acceptable carrier or excipient are also provided.

V. Method of Sequencing Entire Lyssavirus Genome

To facilitate sequencing of the full length ERA genome, a method forsequencing a full length negative strand RNA virus was developed. Thismethod is applicable to the sequencing of a lyssavirus, such as a rabiesvirus, as well as other negative strand RNA viruses. Rabies virus is asingle negative stranded RNA virus with a genome around 12 kb, with therange between 11,918 (Australian bat lyssavirus) and 11,940 (Mokolavirus) bases. The available rabies viral nucleic acid sequences inGENBANK mainly focus on the sequences that encode proteins—nucleocapsidprotein (N), glycoprotein (G), phosphoprotein (P) and matrix protein (M)genes, which are close to the 3′ end of the genome. Prior phylogeneticanalysis is mostly based on N and G genes. But, for remotely relatedrabies viral strains, RNA dependent RNA polymerase (L) gene is the mostsuitable candidate for phylogenetic analysis. Unfortunately, few L genesequences are available in public gene databases. In addition, it hasbeen proposed that both leader and trailer regions at rabies viraltermini play very important roles for (regulation of) viraltranscription and replication. These could be the conserved regions fornucleoprotein encapsidation or the binding sites for L/P proteins, forinstance. Also the inter-genic regions among leader-N, N-P, P-M, M-G,pseudo-gene region and G-L serve as the signals for initiation of viraltranscription. Thus, not only coding regions, but also non-codingregions within the viral genome, could be applied to phylogeneticanalysis or evolution research. These sequences can all more readily beanalyzed using the whole-genome sequencing methods provided herein.

The method includes a single step reverse transcription and a two stepcloning into a suitable vector. This method produces a readily sequencedgenome in the vector, without the need to perform error-prone repeatedRT-PCR reactions. Exploiting the inverted repeat found at the ends ofthe rabies genome (and the genomes of other lyssaviruses), universalprimers have been designed and are described herein for use in the rapidfull-genome sequencing procedures described herein.

The leader and trailer regions in rabies virus contain signals for viraltranscription and replication. Based on analysis the genome sequenceavailable from GenBank, the terminal 11 nucleotides are strictlyconserved in rabies viruses or rabies-related viruses, including Mokolavirus. The rationale for the sequencing methods provided herein is basedon the terminal 11 complementary nucleotides. Because these two 11nucleotide sequences are complementary, they could are not used in thefollow-up PCR reactions. It will be understood that other viruses withinverted repeats can similarly be amplified using primers correspondingto the sequences of those repeats. The 11 antigenome sense nucleotideswere designed as reverse transcription primers for the purified ERAgenome, whose integrity was verified by size comparison and Northernblots. The whole genome cDNA was also confirmed by Northern blot with N,P, M, G, L gene probes and 11 nucleotides as an oligonucleotide probe,which only bound genomic RNA, not viral mRNA.

It is reasonably feasible to reverse transcribe rabies viral wholegenome in one reaction, using carefully designed conserved terminalsequence-corresponding primes, provided the quality of the viral genomepreparation is high.

The sequence of the ERA is closely related to that of SAD, which is oneof its derivatives. This is not surprising, because ERA was sent fromCDC in the 1970s to Switzerland, where researchers adapted it to grow incells, before sending it to Germany, where it was further derived, andthe derivative fully sequenced in ˜1990. Until now, rabies andrabies-related viruses have been classified into seven different types:classic rabies virus type1 (ERA is included), type2 (Lagos bat), type3(Mokola), type4 (Duvenhage), type5 (European bat lyssavirus [EBL] I,type6 (EBL II) and type7 (Australian bat virus) according to serum crossprotection and genetic studies. Sequence analysis plays an importantrole in phylogenetics, evolution research, gene function predictivestudies and other related areas, including locating viral transcriptionand replication regulatory regions, and hence bioinformatics towardspotential therapeutic drugs.

With the development of techniques in reverse transcriptional polymerasechain reaction (RT-PCR), which are known to those of ordinary skill inthe art, now it is relatively easy to reverse transcribe as much as 12kb or more of RNA to cDNA in one reaction. Under optimized conditions,PCR can amplify targets of more than 30 kb in one reaction.

With the provision herein of methods for generating full-length virusgenome sequences, in particularly rabies genome sequences, it nowbecomes practical to analyze differ strains of virus. Effective designof attenuated virus, for instance for use in immunization or productionof immune stimulatory compositions and vaccines, is also enabled usingthe resultant full length genomes.

There is no “general” rabies virus genome, but these genomes arerelated. The similarities range from 60% to 100% in different types.Some regions, such as the L gene, seem more conserved, whereas others,such as the psi region, which does not code for a polypeptide, are morevariable. Not only will rabies and rabies related viruses drift, butalso any RNA viruses will change over time. How the viruses adapt andemerge, is an open question. For this reason, whole genome sequenceanalysis is important for evolutionary, pathogenicity and gene functionstudies.

This system described herein is the first for rabies virus as concernswhole genome sequencing. It is believed to be suitable for other RNAviruses, particularly in the lyssavirus genus. At present, for rabiesvirus phylogenetic studies, scientists only make use of the N, P, or Ggenes, which are most abundant in the infected cells or tissues. It isknown that for remote strain comparison, the L gene comprising more thanhalf of the genome may be an ideal candidate site, which should be used.Unfortunately, such evolutionary comparisons are not possible due to thevery limited data available, let alone the whole genome sequence. Alsofor viral transcription and replication studies, it is supposed that theleader and trailer regions located at the 3′ and 5′ extremities of thegenome play important roles. The inter-genic regions are also thesignals for viral trans and cis studies. All these data are quitelimited, because they are not included in the mRNA. Only the wholegenome sequence can provide the necessary information at this level.Whole genome sequencing is useful not only for vaccine development, itis also applicable for basic virus transcription and replicationstudies. It is also applicable for development siRNA and gene therapy aswell.

VI. ERA Genome Sequence

Using the method described herein, the unique sequence of the ERA rabiesvirus genome has been generated. This sequence is shown in SEQ ID NO: 1.The five proteins of the ERA rabies virus (SEQ ID NOs: 2-6) are encodedat the following positions of the genome: N, 71-1423; P, 1511-2407; M,2491-3104; G, 3318-4892; and L, 5418-11801. The homology between ERA andSAD-B19 are: N 99.56%, P 98.65%, M 96.53%, G 99.05% and L 99.20%,respectively. One specific difference between ERA and SAD-B 19 is theintergenic region between G and the pseudo-gene, with the SAD-B19 Gtranscription stop/polyadenylation signal destroyed.

The ERA rabies virus whole genome sequence is the prerequisite forvaccine development and pathogenicity studies using reverse genetics

VII. Optimized System for Production of Virus

Examples 6 and 7 provide an optimized set of conditions for ERA virusproduction, in which titers reach as high as 10¹⁰ ffu per ml. Inbioreactors, the recovered virus can grow to ˜10⁹ to 10¹⁰ ffu/ml. Suchhigh levels of production are of paramount importance for oral vaccinedevelopment, so sufficient vaccine material can be produced in areasonable amount of time with reasonable resource allocation.

The provided growth conditions can stably produce such high virus titerfor both parental and recombinant ERA strains. These production data arevery important for potential rabies oral vaccine development.

VIII. BSR-G Cell Line for Production of -G Virus

Although strains of RV with deletions of the G protein have beenpreviously rescued from BHK cells, this was not possible with ERA strainvirus lacking the G protein. After inoculation of mice intracerebrallyor intramuscularly with ERA-G, no mice died or showed any rabiessymptoms. The ERA-G (without glycoprotein) can only grow in cells withthe supplementation of the glycoprotein. Otherwise, the mutated viruscannot spread. To help ERA-G grow, a BSR-G cell line was established,which constitutively expressed ERA glycoprotein. Production of this cellline is described in the Examples below. This cell line is useful forrecovery of RV strains such as ERA-G that are refractory to recovery inthe absence of G, as well as for optimizing recovery of other strains.

IX. Reverse Genetics System for Engineering Rabies Virus Vaccines andExpression of Heterologous Proteins

RNA cannot readily be manipulated directly by molecular biologicalmethods. Traditional RNA virus vaccines are from naturally attenuatedisolates, which are difficult to control and provide unpredictableresults. Reverse genetics technology makes it possible to manipulate RNAviruses as DNA, which can be mutated, deleted or reconstructed accordingto deliberate designs. Every gene function can be studied carefully,independently, and in concert, which benefits vaccine development.Reverse genetics involves reverse transcription of the RNA viral genomeinto cDNA, and cloning into a vector, such as a plasmid. Aftertransfection of host cells, the vector is transcribed into RNA, to beencapsidated by structural proteins, which can also be supplied byplasmids. The encapsidated RNA forms a ribonucleoprotein complex, whichresults in virions that can be recovered.

Although three systems for rabies virus (RV) reverse genetics have beenpublished (Schnell et al., The EMBO J. 13, 4195-4203, 1994; Inoue etal., J. Virol. Method. 107, 229-236, 2003; Ito et al., Microbiol.Immunol. 47, 613-617, 2003), these systems are not readily adaptable toother strains. At present, no rabies virus strain has been recoveredwith the aid of helper plasmids from a different strain, even when thestrains are closely related. Thus, for any specific virus strainmutation or vaccine development, a specifically tailored system must bedeveloped.

The ERA strain is a good candidate for rabies oral vaccine development,but its residual pathogenicity is obvious. During the 1970s, the ERA RVunderwent extensive vaccine development (Black and Lawson, Can. J. Comp.Med. 44:169-176, 1980; Charlton, and Casey, Can. J. Vet. Res.20:168-172, 1978; Lawson, and Crawley, Can. J. Vet. Res. 36: 339-344,1972). Both ERA and SAD-B19 are derived from SAD. In primary oralvaccine trials, SAD-B 19 was effective in both raccoons and skunks,while ERA was not. Additionally, ERA kills two-week old miceadministrated intra cerebrally (i.c.), as demonstrated in animal tests.These observations raise questions of the relationship between these twoRV strains and the potential effects of subtle alterations. From fullviral genome sequence comparison, ERA and SAD-B 19 share extremely highnucleotide identity and amino acid homologies. To clarify the geneticbasis of immunogenicity and pathogenicity of these highly relatedstrains of rabies virus, an efficient reverse genetics system wasdeveloped for ERA, which differs from reverse genetics systemspreviously reported for rabies virus.

The rabies reverse genetics system disclosed herein is useful for avariety of purposes, including: (1) to attenuate ERA virus in a definedmanner for vaccine development; (2) to produce ERA virus vectors forexpressing heterologous ORFs (e.g., in the context of therapeuticcompositions, such as vaccines and gene therapy); (3) to define thegenetic basis of ERA RV pathogenesis; and (4) to determine thebiological effects of genetic differences between the ERA and SADviruses.

The reverse genetics system has some or all of the followingcharacteristics, illustrated schematically in FIG. 1A using theexemplary ERA strain antigenomic cDNA.

This system is based a full length transcription plasmid plus aplurality of helper plasmids (e.g., five helper plasmids). The helperplasmids encode the N, P, L proteins, and optionally the G protein, aswell as the T7 polymerase. Although the G protein is not necessary forvirus rescue, it improves virus recovery efficiency or virus buddingwhen included in transfection.

Transcription involves both cellular RNA dependent RNA polymerase II,which is available in mammalian cells, and T7 RNA polymerase, which issupplied by pNLST7 plasmids. The dual polymerases result in virusrecovery efficiency is both high and stable.

In the transcription plasmid, hammerhead and hepatitis delta virusribozymes flank a rabies virus (e.g., ERA strain) antigenomic cDNA,enabling the production of authentic 5′ and 3′ ends of antigenomic vRNAby transcription. The first ten nucleotides of the hammerhead sequenceare designed to be complementary to the first ten nucleotides of theantisense genomic sequence. For example, the first ten nucleotides ofthe hammerhead sequence for the ERA antigenomic cDNA are:

TGTTAAGCGT. (SEQ ID NO: 19)

Two modified T7 RNA polymerase constructs have been established, whichsupport virus recovery more efficiently than the wild type T7 RNApolymerase applied previously. One T7 RNA polymerase has been mutatedfrom the first ATG to AT. The second T7 RNA polymerase has an eightamino acid nuclear localization signal (NLS) derived from the SV40 viruslarge T antigen fused after the first ATG from the parental T7: ATG CCAAAA AAG AAG AGA AAG GTA GAA (SEQ ID NO: 20). The NLS is underlined.Addition of the NLS results in the T7 RNA polymerase being presentpredominantly in the nucleus. Following transfection mechanism of theNLS modified plasmid, the DNA/transfection reagent complex binds tosurface of the cell. Through endocytosis, the complex is taken into theendosome/lysosome, and the DNA is released into the cytosol. In theabsence of the NLS, the majority of the transfected plasmids areretained in the cytosol and only a small percentage of the released DNAreaches the nucleus, where it is transcribed into RNA. After proteinsynthesis, the NLST7 RNA polymerase is transported back to the cellnucleus, and the helper plasmids (with T7/CMV promoters) in the nucleuswill be transcribed by both NLST7 and cellular polymerase II. Thus, moremRNAs of the helper plasmids and cRNA of the full-length pTMF or itsderivatives were synthesized and resulted in high efficiency of virusrecovery.

After the initial expression of NLST7 by CMV promoter, NLST7 polymerasebinds to pT7 for transcription of NLST7 gene. Through modification ofthe transcripts in the nucleus, more NLST7 mRNA is synthesized,resulting in more expression of NLST7 polymerase. The pT7 of the NLST7polymerase as well as of the full length antigenomic transcription unitis under the control of the NLST7 polymerase, which acts as an“autogene.” The autogene mechanism of NLST7 RNA polymerase isillustrated in FIG. 2. After expression of T7 RNA polymerase in thenucleus, the transfected T7 constructs continue to transcribe fulllength RNA template for N protein encapsidation and/or L proteinbinding, enhancing virus recovery efficiency.

The T7 polymerase, and all other plasmids, except the N protein encodingplasmid pTN, are placed under control of both CMV and T7 transcriptionalregulatory elements. The N protein encoding nucleic acid is under thecontrol of a T7 promoter and is translated in cap-independent mannerbased on an IRES (Internal Ribosome Entry Site). Cellular RNA polymeraseII alone can help the recovery of RV if all the plasmids were clonedunder the control of the CMV promoter (19). In the ERA reverse geneticssystem disclosed herein, only pTN is under the control of the T7promoter and was translated in a cap-independent manner. All otherconstructs are under control of both CMV and the T7 transcriptionalregulatory elements. Typically, in RV, N synthesis is abundant and theratio among N, P and L is approximately 50:25:1. To mimic the wild typeviral transcription and assembly in RV reverse genetics, N expressionshould be the highest. With the aid of NLST7 polymerase and IREStranslation mode, N protein was expressed efficiently after plasmidtransfection. This reduces competition for transcription with housekeeping genes in host cells, because the T7 transcription initiationsignal does not exist in mammalian cells, and results in increasedefficiency of T7 transcription.

To enhance production of viral proteins, the helper plasmids can beconstructed to incorporate a Kozak sequence that has been optimized forthe translation efficiency for each protein encoding sequence. Exemplaryoptimized Kozak sequences are shown in Table 2.

TABLE 2 Optimized Kozak sequences. constructs promoters Kozak contextSEQ ID NO: Special characters pTMF CMV/T7 n/a n/a HamRZ/HdvRZ at endspTN T7/IRES ACCACCATGG SEQ ID NO: 21 n/a pMP CMV/T7 ACCACCATGASEQ ID NO: 22 n/a pMG CMV/T7 ACCACCATGG SEQ ID NO: 21 n/a pML CMV/T7ACCACCATGC SEQ ID NO: 23 n/a pNLST7 CMV/T7 ACCACCATGA SEQ ID NO: 228 amino acids NLS CMV/T7 symbolizes the CMV promoter ahead of a pT7promoter. The HdRz indicates a hammerhead ribozyme and HDVRz is thehepatitis delta virus ribozyme. The pTMF is the full-lengthtranscription plasmid, and the pTN, pMP, pMG, pML and pNLST7 are helperplasmids.

After five days post-transfection in the ERA reverse genetics system,the rescued virus reliably and repeatedly grew to 10⁷ ffu/ml withoutfurther amplification.

X. Derivative Viruses

The complete mechanism of Rabies virus pathogenicity has not been fullycharacterized, making rational vaccine design problematic. For example,the RV glycoprotein appears to play a role both in pathogenicity andimmunogenicity of rabies virus. Mutations (such as at position 333 ofthe glycoprotein) result in virus that does not cause lethal infectionin adult mice (Ito et al., Microbiol. Immunol. 38, 479-482, 1994; Ito etal., J. Virol. 75, 9121-9128, 2001). However, overexpression of RVglycoprotein has been shown to lead to the enhancement of apoptosis andantiviral immune response (Faber et al., J. Virol. 76, 3374-3381, 2002).Thus ERA strain virus with a modified (for example, deleted, amino acidsubstituted) G protein could be a particular strain for vaccinedevelopment.

Recombinant rabies viruses with favorable properties can be designedusing the reverse genetics system disclosed herein. Exemplaryrecombinant viruses disclosed herein include, in addition to theparental ERA strain, ERA without Psi-region (ERA-), ERAgreen1 (greenfluorescent gene inserted in the Psi-region), ERAgreen2 (greenfluorescent gene cloned at P-M intergenic region), ERA2g (containing anextra copy of G in Psi-region), ERAg3 (G mutated at 333 amino acid),ERA2g3 (containing an extra copy of mutated G in Psi-region), ERAgm (Mand G genes switched in the genome), and ERAgmg (two copies of G in therearranged ERAgm construct). These exemplary strains are illustratedschematically in FIG. 3.

Modified strains having deleted and/or mutated glycoproteins areparticularly suited for use as immunogenic compositions for pre and postexposure treatment of rabies virus because such viruses are incapable ofspreading between cells and causing disease. Additionally, modifiedviruses such as ERA2g3, which overexpresses the G protein due to aduplication of sequences encoding a mutated glycoprotein is predictedenhance apoptosis and elicit an increased anti-viral immune response.

For example, after intracerebral and intramuscular inoculation of micewith a deletion of G (ERA-G), no adverse events were observed. Moreover,the ERA-G protected mice from lethal challenge by a street RV strain.Thus, ERA-G appears to be a safer strain that ERA for vaccinedevelopment. Additionally, mutation of arginine at amino acid position333 of the ERA G to glutamic acid (from nucleotides AGA to GAG, as inthe ERAg3 and ERA2g3 strains) results in an attenuated virus.Attenuation was confirmed via animal inoculation tests. Becauseoverexpression of RV G results in the enhancement of apoptosis andantiviral immune responses, attenuated viruses such as ERA2g3 thatpossess multiple copies of G are particularly favorable as vaccinecandidates.

The system for rabies vaccine development described herein is notlimited to modifications of the G gene, but is similarly applicable toeach of the viral proteins. To facilitate a systematic approach tomodifying the various protein components, detailed mapping ofpathogenicity can be solved by reverse genetics based on the sequencedata presented herein.

The reverse genetics system described herein also enables a rabies virusvector system for foreign (heterologous) gene expression. The described,non-limiting embodiment is based on the ERA virus. An extratranscription unit is shown herein to be functional in two differentlocations after incorporation into the ERA RV genome. In one embodiment,an extra transcription unit is incorporated in the position of the psiregion (trans 1). In an alternative embodiment, an extra transcriptionunit is inserted into the RV P-M intergenic region.

In single stranded negative RNA viruses, the 3′-distal sequences of thegenome serve mainly as a transcription promoter, while the 5′-terminalsequences of the genome serve as a replication promoter (Conzelmann andSchnell, J. Virol. 68:713-719, 1994; Finke et al., J. Virol.71:7281-7288, 1997). Thus, trans2 occupies a position that results instronger transcription for driving ORFs expression than trans1. Thus,the vectors disclosed herein can be used to modulate expression of aheterologous ORF to a desired level, simply by selecting the positioninto which the ORF is inserted in the vector. For example, when a highlevel of expression of a protein is desired, the trans 2 is typically anideal position for the insertion of the heterologous ORF. Similarly, ifmore moderate levels of expression are desired, the heterologous ORF canbe inserted into trans 1. Optimal expression levels for each ORF and forparticular applications can be determined by one of skill in the artwithout undue experimentation.

Thus, the viral vectors provided herein are excellent construct forforeign gene insertion and expression, as is demonstrated herein withrespect to expression of the green fluorescent protein gene. Althoughthe utility and efficacy of the disclosed vectors is demonstrated withrespect to GFP, it should be noted that the vectors are equally suitablefor expressing any gene or ORF of interest.

As noted, the rabies-based heterologous expression system providedherein can be used to express any foreign (heterologous) protein(s). Itis particularly contemplated, by way of example, that such heterologousgenes are from another pathogenic organism, such as other pathogenicviruses, for instance SARS virus, Nipah virus, etc. In addition, thedisclosed vectors can be used for delivery of other therapeutic genes,including for example, that encode proteins of therapeutic value orfunctional RNA molecules, such as siRNAs.

XI. Pharmaceutical and Immune Stimulatory Compositions and Uses Thereof

Pharmaceutical compositions including attenuated or fixed rescuedviruses, virus nucleic acid sequences or virus polypeptides comprisingat least one virus epitope are also encompassed by the presentdisclosure. These pharmaceutical compositions include a therapeuticallyeffective amount of one or more active compounds, such as an attenuatedor fixed virus, a virus polypeptides comprising at least one virusepitope, or one or more nucleic acid molecules encoding thesepolypeptides, in conjunction with a pharmaceutically acceptable carrier.It is contemplated that in certain embodiments, virus nucleic acidsequences or virus polypeptides comprising multiple virus epitopes willbe useful in preparing the pharmaceutical compositions of thedisclosure.

Disclosed herein are substances suitable for use as immune stimulatorycompositions for the inhibition or treatment (either pre-exposure orpost-exposure) of a virus infection, for example, a rabies virusinfection.

In one embodiment, an immune stimulatory composition contains anattenuated or fixed rescued (recombinant) virus. In another embodiment,the composition contains an isolated or recombinant virus polypeptideincluding at least one virus epitope (such as a rabies virus G protein).In a further embodiment, the immune stimulatory composition contains anucleic acid vector that includes at least one virus nucleic acidmolecule described herein, or that includes a nucleic acid sequenceencoding at least one virus epitope. In a specific, non-limitingexample, a nucleic acid sequence encoding at least one virus epitope isexpressed in a transcriptional unit, such as those described inpublished PCT Application Nos. PCT/US99/12298 and PCT/US02/10764 (bothof which are incorporated herein in their entirety).

The immune stimulatory viruses, virus polypeptides, constructs orvectors encoding such polypeptides, are combined with a pharmaceuticallyacceptable carrier or vehicle for administration as an immunestimulatory composition to human or animal subjects.

The immunogenic formulations may be conveniently presented in unitdosage form and prepared using conventional pharmaceutical techniques.Such techniques include the step of bringing into association the activeingredient and the pharmaceutical carrier(s) or excipient(s). Ingeneral, the formulations are prepared by uniformly and intimatelybringing into association the active ingredient with liquid carriers.Formulations suitable for parenteral administration include aqueous andnon-aqueous sterile injection solutions which may contain anti-oxidants,buffers, bacteriostats and solutes which render the formulation isotonicwith the blood of the intended recipient; and aqueous and non-aqueoussterile suspensions which may include suspending agents and thickeningagents. The formulations may be presented in unit-dose or multi-dosecontainers, for example, sealed ampoules and vials, and may be stored ina freeze-dried (lyophilized) condition requiring only the addition of asterile liquid carrier, for example, water for injections, immediatelyprior to use. Extemporaneous injection solutions and suspensions may beprepared from sterile powders, granules and tablets commonly used by oneof ordinary skill in the art.

In certain embodiments, unit dosage formulations are those containing adose or unit, or an appropriate fraction thereof, of the administeredingredient. It should be understood that in addition to the ingredientsparticularly mentioned above, formulations encompassed herein mayinclude other agents commonly used by one of ordinary skill in the art.

The compositions provided herein, including those for use as immunestimulatory compositions, may be administered through different routes,such as oral, including buccal and sublingual, rectal, parenteral,aerosol, nasal, intramuscular, subcutaneous, intradermal, and topical.They may be administered in different forms, including but not limitedto solutions, emulsions and suspensions, microspheres, particles,microparticles, nanoparticles, and liposomes.

The volume of administration will vary depending on the route ofadministration. By way of example, intramuscular injections may rangefrom about 0.1 ml to about 1.0 ml. Those of ordinary skill in the artwill know appropriate volumes for different routes of administration.

A relatively recent development in the field of immune stimulatorycompounds (for example, vaccines) is the direct injection of nucleicacid molecules encoding peptide antigens (broadly described in Janeway &Travers, Immunobiology: The Immune System In Health and Disease, page13.25, Garland Publishing, Inc., New York, 1997; and McDonnell & Askari,N. Engl. J. Med. 334:42-45, 1996). Vectors that include nucleic acidmolecules described herein, or that include a nucleic acid sequenceencoding a virus polypeptide comprising at least one virus epitope maybe utilized in such DNA vaccination methods.

Thus, the term “immune stimulatory composition” as used herein alsoincludes nucleic acid vaccines in which a nucleic acid molecule encodinga virus polypeptide comprising at least one virus epitope isadministered to a subject in a pharmaceutical composition. For geneticimmunization, suitable delivery methods known to those skilled in theart include direct injection of plasmid DNA into muscles (Wolff et al.,Hum. Mol. Genet. 1:363, 1992), delivery of DNA complexed with specificprotein carriers (Wu et al., J. Biol. Chem. 264:16985, 1989),co-precipitation of DNA with calcium phosphate (Benvenisty and Reshef,Proc. Natl. Acad. Sci. 83:9551, 1986), encapsulation of DNA in liposomes(Kaneda et al., Science 243:375, 1989), particle bombardment (Tang etal., Nature 356:152, 1992; Eisenbraun et al., DNA Cell Biol. 12:791,1993), and in vivo infection using cloned retroviral vectors (Seeger etal., Proc. Natl. Acad. Sci. 81:5849, 1984). Similarly, nucleic acidvaccine preparations can be administered via viral carrier.

The amount of immunostimulatory compound in each dose of an immunestimulatory composition is selected as an amount that induces animmunostimulatory or immunoprotective response without significant,adverse side effects. Such amount will vary depending upon whichspecific immunogen is employed and how it is presented. Initialinjections may range from about 1 μg to about 1 mg, with someembodiments having a range of about 10 μg to about 800 μg, and stillother embodiments a range of from about 25 μg to about 500 μg. Followingan initial administration of the immune stimulatory composition,subjects may receive one or several booster administrations, adequatelyspaced. Booster administrations may range from about 1 μg to about 1 mg,with other embodiments having a range of about 10 μg to about 750 μg,and still others a range of about 50 μg to about 500 μg. Periodicboosters at intervals of 1-5 years, for instance three years, may bedesirable to maintain the desired levels of protective immunity.

It is also contemplated that the provided immunostimulatory moleculesand compositions can be administered to a subject indirectly, by firststimulating a cell in vitro, which stimulated cell is thereafteradministered to the subject to elicit an immune response. Additionally,the pharmaceutical or immune stimulatory compositions or methods oftreatment may be administered in combination with other therapeutictreatments.

The preparation of food-baits containing immune stimulatory compositionsis also within the ordinary skill of those in the art. For example, thepreparation of food-baits containing live RV vaccines is disclosed inWandeler et al. (Rev. Infect. Dis. 10 (suppl. 4):649-653, 1988), Aubertet al. (pp. 219-243, in Lyssaviruses (Rupprecht et al., eds.),Springer-Verlag, New York, 1994), and Fu et al. (pp. 607-617, in NewGeneration Vaccines (2^(nd) Edit.) (Levine et al., eds.), Marcel Dekker,Inc., New York, 1997), the entire disclosures of each of which areincorporated by reference herein.

XII. Kits

Also provided herein are kits useful in the detection and/or diagnosisof virus infection, for instance infection with a rabies virus or otherlyssavirus. An example of an assay kit provided herein is a recombinantvirus polypeptide (or fragment thereof) as an antigen and anenzyme-conjugated anti-human antibody as a second antibody. Examples ofsuch kits also can include one or more enzymatic substrates. Such kitscan be used to test if a sample from a subject contains antibodiesagainst a virus-specific protein. In such a kit, an appropriate amountof a virus polypeptide (or fragment thereof) is provided in one or morecontainers, or held on a substrate. A virus polypeptide can be providedin an aqueous solution or as a freeze-dried or lyophilized powder, forinstance. The container(s) in which the virus polypeptide(s) aresupplied can be any conventional container that is capable of holdingthe supplied form, for instance, microfuge tubes, ampoules, or bottles.

The amount of each polypeptide supplied in the kit can be anyappropriate amount, and can depend on the market to which the product isdirected. For instance, if the kit is adapted for research or clinicaluse, the amount of each polypeptide provided would likely be an amountsufficient for several assays. General guidelines for determiningappropriate amounts can be found, for example, in Ausubel et al. (eds.),Short Protocols in Molecular Biology, John Wiley and Sons, New York,N.Y., 1999 and Harlow and Lane, Using Antibodies: A Laboratory Manual,CSHL, New York, 1999.

The following examples are provided to illustrate certain particularfeatures and/or embodiments. These examples should not be construed tolimit the invention to the particular features or embodiments described.

EXAMPLES Example 1 Sequencing of ERA RV

This example provides a description of a method for sequencing the fulllength genome of a rhabdovirus, particularly in this case a rabiesvirus.

Rabies virus strain ERA was obtained from the CDC archive and waspropagated in baby hamster kidney (BHK-21) cells. Virus was harvestedafter four days infection at 37° C., in a 5% CO₂ incubator and waspurified. Briefly, the cell supernatant was collected and centrifuged at2,000 rpm for 15 minutes to remove the cell debris. The clearsupernatant was subjected to further centrifugation at 18,000 rpm for 1hour. The pellet was resuspended in PBS and subjected to rabies genomicRNA extraction.

Total RNA from ERA-infected BHK-21 cells was extracted with Trizol™reagent (GIBCO Invitrogen) according to the protocol recommended by themanufacturer. ERA genomic RNA was purified from the concentrated ERAvirus supernatant with a high pure viral RNA kit from Roche.

Integrity of the purified ERA genomic RNA was verified by gelelectrophoresis and Northern blot by N, P, G, and M hybridizationprobes. Briefly, 5 μg of genomic RNA was loaded in a denatured RNA geland transferred to a nylon membrane for hybridization. The probe waslabeled using the Dig DNA labeling kit from Roche, according tomanufacturer's instructions.

The 11 conserved nucleotides from the rabies virus 5′ antigenome weredesigned as a primer for reverse transcription. The RT reaction wascarried out with a first-strand cDNA synthesis kit from Invitrogen. Thecomplete cDNA from the ERA genome was confirmed by Northern blot usingN, P, M, and G probe hybridization, as well as the 11 conservednucleotides as oligonucleotide probes labeled by Digoxin.

Two sets of primers were chosen for PCR reactions, which amplify thewhole ERA genome in two contiguous fragments. One set of primers iscomposed of the 11 nucleotides at 5′ antigenome end, Le5: ACGCTTAACAA(SEQ ID NO: 24) and BLp3: GTCGCTTGCTAAGCACTCCTGGTA (SEQ ID NO: 25).Another set contains the 11 complementary nucleotides at the 5′ genomeend, Le3: TGCGAATTGTT (SEQ ID NO: 26) and BLp5 CCAG GAGTGCTTAGCAAGCGACCT(SEQ ID NO: 27). The Blp3 and Blp5 primers are located in a relativelyconserved region in the rabies virus genome.

PCR fragments were purified and cloned into the TOPO vector purchasedfrom Invitrogen. Sequencing was conducted in an ABI 310 sequencer andthe sequence was assembled by BioEdit™ software or SeqMerge™ softwarefrom Accelrys in the GCG environment.

The complete aligned sequence of the ERA genome is provided in SEQ IDNO: 1. The positions of individual protein encoding sequences areprovided in Table 3, with reference to SEQ ID NO: 1. The amino acidsequences of the N, P, M, G and L proteins are provided in SEQ ID NOs: 2through 6, respectively.

TABLE 3 Positions of protein encoding sequences of rabies virus ERAstrain Positions in rERA Gene/genome NT sequence ERA 11930   1-11930 N1412  71-1423 P 962 1511-2407 M 789 2491-3104 G 1647 3318-4892Psi-region 398 L 6445  5418-11801 Leader 58 Trailer 70

This method can be used for both rabies and rabies-related viruses.Rabies and rabies-related viruses have at least seven putative speciestypes. The provided sequence method can be used also for other negativestranded RNA viruses. This is because almost all the negative-strandedRNA virus genomes have approximately 12 conserved nucleotides at bothdistal ends, which similarly can serve as primers for RT-PCR. Theprimers will of course be different for different viral species, and thesequence of specific primers can be determined by one of ordinary skillbased on the teachings herein.

Example 2 Construction of Plasmids for a Reverse Genetics System forRabies Virus

This example describes the design and development of a Reverse GeneticsSystem for Rabies Virus. Rabies virus strain ERA was obtained from theATCC and was prepared as described (Wu et al., J. Virol. 76, 4153-4161,2002). To obtain virus genome full-length virus cDNA, BSR cells (a cloneof baby hamster kidney, BHK, cells) were infected with ERA strain virusand grown in Dulbecco's minimal essential medium supplemented with 10%of fetal bovine serum. Supernatants were recovered and subjected tocentrifugation at 22,000 g for 1 hour. The virus pellets were collectedfor viral genomic RNA purification by use of a RNA virus extraction kitpurchased from Qiagen (Valencia, Calif.) according to the manufacturer'sinstructions. The integrity of viral genomic RNA was confirmed by gelelectrophoresis. Viral genomic cDNA was transcribed with thefirst-strand cDNA synthesis kit from Invitrogen (Carlsbad, Calif.). Thereverse transcription (RT) reaction mixture was applied to amplificationby the polymerase chain reaction (PCR) for the synthesis of full-lengthviral genomic cDNA, N, P, G and L genes, respectively. For assemblingthe full-length virus genomic cDNA, a pTMF plasmid was constructed infour sequential steps as illustrated schematically in FIG. 1B.Superscript III reverse transcriptase and proof reading platinum pfxpolymerase (Invitrogen, Carlsbad, Calif.) were applied for cDNAtranscript synthesis and consecutive PCR amplifications. For reversetranscription reactions, 1 μg of purified genomic RNA was used in the RTreaction mix and incubated at 50° C. for 80 min, followed by heating at85° C. for 5 minutes to inactivate Superscript III. After the RTreaction, 1 unit of RNaseH was added to digest template RNA in thecDNA-RNA hybrids.

To generate full-length virus genomic cDNA, two overlapping fragmentswere amplified by RT-PCR as follows: Fragment1 (F1) was RT-PCR amplifiedwith primers: Le5-Kpn (CCGGGTACCACGCTTAAC AACCAGATCAAAGA; SEQ ID NO: 28,Kpn1 recognition site underlined) and Le3-Blp(TAGGTCGCTTGCTAAGCACTCCTGGTAGGAC; SEQ ID NO: 29, Blp1 recognition siteunderlined). Fragment 2 (F2) was RT-PCR amplified with primers: Tr5-Blp(GTCCTACCAGGAGTGCTTAGCAAGCGACCTA; SEQ ID NO: 30, Blp1 recognition siteunderlined) and Tr3-Pst (AAAACTGCAGACGCTTAACAAATAAACAACAAAA; SEQ ID NO:31, Pst1 recognition site underlined). After successful synthesis of theabove two fragments, F1 digested by Kpn1 and Blp1 restriction enzymeswas subjected to gel purification and cloned to pBluescriptIISK(+)phagemid (Stratagene, La Jolla, Calif.) to form the pSKF1 plasmid. Thegel purified F2 fragment, cut by Blp1 and Pst1 was consecutively clonedto the pSKF1 plasmid to form the full-length viral antigenomic cDNA.Hammerhead ribozyme (oligo1,CAAGGCTAGCTGTTAAGCGTCTGATGAGTCCGTGAGGACGAAACTATAGGAAAGGAATTCCTATAGTCGGTACCACGCT; SEQ ID NO: 32, Nhe1 and Kpn1 recognitionsites underlined; Oligo2,AGCGTGGTACCGACTATAGGAATTCCTTTCCTATAGTTTCGTCCTCACGGACTCATCAGACGCTTAACAGCTAGCCTTG; SEQ ID NO: 33, Kpn1 and Nhe1 recognitionsites underlined) was synthesized containing a Nhe1 recognition site atthe 5′ end and a Kpn1 site at the 3′ end. This was fused ahead of the 5′end of the F1 fragment. A hepatitis delta virus ribozyme (oligo3,GACCTGCAGGGGTCGGCATGGCATCTCCACCTCCTCGCGGTCCGACCTGGGCATCCGAAGGAGGACGCACGTCCACTCGGATGGCTAAGGGAGGGCGCGGC CGCACTC; SEQ ID NO:34, Pst1 and Not1 recognition sites underlined; Oligo4,GAGTGCGGCCGCGCCCTCCCTTAGCCATCCGAGTGGACGTGCGTCCTCCTTCGGATGCCCAGGTCGGACCGCGAGGAGGTGGAGATGCCATGCCGACCCCTGC AGGTC; SEQ ID NO:35, Not1 and Pst1 recognition sites underlined) (Symons, Annu. Rev.Biochem. 61: 641-671, 1992) was synthesized, having a Pst1 site at its5′ end and a Not1 site at its 3′ end, and was fused to the 3′ end of theF2 fragment. The connective Kpn1 recognition site, between thehammerhead ribozyme and the F1 fragment, and the Pst1 site between theF2 fragment and the hepatitis delta virus ribozyme, were deleted bysite-directed mutagenesis. The full-length viral antigenomic cDNA wassandwiched by the hammerhead and hepatitis delta virus ribozymes. Thiswas removed and cloned to the pBluescriptIISK(+) phagemid to make a pSKFconstruct. The full viral antigenomic cDNA with two ribozymes was fuseddownstream of the T7 transcription initiation site under control of theCMV immediate-early promoter in pcDNA3.1/Neo (+) plasmid (Invitrogen,Carlsbad, Calif.). This last step finished the construction of the pTMFplasmid.

The wild type ERA viral genome includes a polyA tract of eight residues(polyA₈) in the intergenic region between the G and Psi regions. Todistinguish the rescued ERA (rERA) virus from the parental strain, astretch of seven A (polyA₇) was introduced to the pTMF construct bydeletion of one A instead of the original polyA₈. After rERA virus wasrecovered, RT-PCR was performed and subsequent sequence data confirmedthe existence of the introduced poly A₇ sequence marker.

pTN plasmid: The N gene was amplified by RT-PCR with primers (5N:ACCACCATGGATGCCGACAAGATTG; SEQ ID NO: 36, Nco1 recognition site andstart codon underlined; and 3N: GGCCCATGGTTATGAGTCACTCGAATATGTCTT; SEQID NO: 37, Nco1 recognition site and stop codon underlined) and clonedto the pCITE-2a(+) (Cap-Independent Translation Enhancer) plasmid(Novagen, Madison Wis.).

pMP plasmid: the P gene was amplified by RT-PCR with primers (5P:TTGGTACCACCATGAGCAAGATCTTTGTCAATC; SEQ ID NO: 38, Kpn1 recognition siteand start codon underlined; and 3P: GGAGAGGAATTCTTAGCAAGATGTATAGCGATTC;SEQ ID NO: 39, EcoR1 recognition site and stop codon underlined) andcloned to the pcDNA3.1/Neo (+) plasmid.

pMG plasmid: the G gene was amplified by RT-PCR with primers (5G:TTGGTACCACCATGGTTCCTCAGGCTCTCCTG; SEQ ID NO: 40, Kpn1 recognition siteand start codon underlined; and 3G: AAAACTGCAGTCACAGTCTGGTCTCACCCCCAC;SEQ ID NO: 41, Pst1 recognition site and stop codon underlined) andcloned to the pcDNA3.1/Neo (+) plasmid.

pML plasmid: the L gene was amplified by RT-PCR with primers (5L:ACCGCTAGCACCACCATGCTCGATCCTGGAGAGGTC; SEQ ID NO: 42, Nhe1 recognitionsite and start codon underlined; and 3L:AAAACTGCAGTCACAGGCAACTGTAGTCTAGTAG; SEQ ID NO: 43, Pst1 recognition siteand stop codon underlined) and cloned to the pcDNA3.1/Neo (+) plasmid.

pT7 plasmid: genomic DNA from bacteria BL-21(Novagene, Madison, Wis.)was extracted with the DNeasy™ Tissue Kit (Qiagen, Valencia, Calif.)according to the manufacturer's instructions. The T7 RNA polymerase genewas amplified from the purified genomic DNA by PCR with primers (5T7:TCGCTAGCACCACCATGAACACGATTAACATCGCTAAG; SEQ ID NO: 44, Nhe1 recognitionsite and start codon underline; and 3T7:GATGAATTCTTACGCGAACGCGAAGTCCGACTC; SEQ ID NO: 45, EcoR1 recognition siteand stop codon underlined) and cloned to the pcDNA3.1/Neo (+) plasmid.

pNLST7 plasmid: an eight amino acid nuclear location signal (NLS),derived from SV40 large T antigen, was added to the N terminus of the T7RNA polymerase by PCR amplification, using the pT7 plasmid as thetemplate, with primers (5T7NLS:TCGCTAGCCACCATGCCAAAAAAGAAGAGAAAGGTAGAAAACACGATTAA CATCGCTAAGAAC; SEQ IDNO: 46, NLS underlined and 3T7 primer). The amplified fragment wasdesignated NLST7, and was cloned to pcDNA3.1/Neo (+) to form the pNLST7construct.

pGFP plasmid: Monster Green Fluorescent Protein (GFP) plasmid phMGFP waspurchased from Promega (Madison, Wis.). The GFP gene was amplified byPCR with primers (GFP5: AAAACTGCAGGCCACCATGGGCGTGATCAAG; SEQ ID NO: 47,Pst1 recognition site and start codon underlined; and GFP3:CCGCTCGGTACCTATTAGCCGGCCTGGCGGG; SEQ ID NO: 48, Kpn1 recognition siteand stop codon underlined) and cloned to the pcDNA3.1/Neo (+) plasmid.

All plasmid constructs were sequenced at least three times to confirmthe absence of unexpected mutations or deletions after cloning,site-directed mutagenesis, or gene deletion. Additionally, presence of amarker sequence consisting of a polyA tract having seven adenosineresidues rather than the eight residues observed in the wild type ERAgenome between the glycoprotein and Psi region was confirmed.

Example 3 T7 RNA Polymerase Expression in BSR Cells

This example demonstrates that addition of a nuclear localization signalto the phage T7 RNA polymerase directs expression of the polymerase inthe nucleus of transfected cells. Transfection of BSR cells wasperformed as described by Wu, et al. (J. Virol. 76, 4153-4161, 2002).Briefly, BSR cells near 80% confluence in a six-well-plate weretransfected with 0.5 μg of pT7 or pNLST7 plasmid per well, respectively.At 48 hours after transfection, cells were fixed with 80% chilledacetone for 1 h and dried at room temperature. Mouse monoclonal antibodyagainst the T7 RNA polymerase and goat anti mouse IgG-FITC conjugatewere successively added, and were washed in the two-step indirectfluorescent staining procedure. Results were recorded after UVmicroscopy. The T7 RNA polymerase expressed from pT7 without a nuclearlocalization signal was observed primarily in the cytosol, whereas NLST7polymerase including a nuclear localization signal was presentpredominantly in the nucleus of cells. These results indicated thataddition of an NLS effectively targeted the T7 RNA polymerase to thenucleus of transfected cells.

Example 4 Establishment of Constitutively Expressed ERA Glycoprotein BSRCell Line

This example describes the design and production of a BHK cell line thatconstitutively expresses the ERA glycoprotein. A BHK cell line thatexpresses the ERA glycoprotein was constructed using the Flp-In™ system(Invitrogen, Carlsbad, Calif.). Briefly, Flp-In™-BHK cells (containing asingle integrated Flp recombination target site) were grown toapproximately 20% confluence in one six-well-plate and maintained incommon DMEM medium, supplemented with 100 μg/ml of Zeocin, beforetransfection. The ERA G gene was amplified by PCR using pMG plasmid astemplate with primers EF5G5 (CACCATGGTTCCTCAGGCTCTCCTG; SEQ ID NO: 49)and EF5G3 (TCACAGTCTGGTCTCACCCCCAC; SEQ ID NO: 50), and cloned to apEF5/FRT/V5-D-TOPO vector (Invitrogen, Carlsbad, Calif.) to create thepEFG construct. The pOG44 plasmid expressing Flp recombinase togetherwith pEFG at the ratio of 10:1 was co-transfected to the Flp-In™-BHKcells. After transfection, the cells were kept in DMEM without Zeocin,but with hygromycin B at 400 μg/ml. After 48 hours, the cells were splitso that no more than 20% confluency occurred the next day. The cellswere grown in hygromycin B selective medium at 37° C. for approximatelyone week. The target ERA G expression was detected by indirectfluorescent staining with human anti-G monoclonal antibody and goatanti-human IgG-FITC conjugate. The cell line constitutively expressingthe G was designated as BHK-G, and was used for the growth of ERA-Gvirus.

Example 5 Defined Modification of Rabies VirusEvelyn-Rokitnicki-Abelseth (ERA) Strain

In addition to the parental ERA virus strain described above, derivativevirus strains were developed using the reverse genetics system disclosedherein. Several exemplary modified viruses were produced, namely ERA-(deletion of the whole psi-region), ERAgreen1 (green florescent proteingene inserted in ERA viral genome psi region), ERAgreen2 (greenflorescent protein gene inserted in phosphoprotein and matrix proteinintergenic region), ERA2g (containing an extra copy of glycoprotein inthe psi-region), ERAg3 (with a mutation at amino acid 333 inglycoprotein), ERA2g3 (with an extra copy of mutated glycoprotein atAa333 in psi-region), ERA-G (with glycoprotein deleted) ERAgm (M and Ggenes switched in the genome), and ERAgmg (two copies of G in therearranged ERAgm construct) These derivatives are illustratedschematically in FIG. 3. By optimizing the growth conditions asdescribed, all of the rescued viruses can be obtained at virus titers of10⁹ to 10¹⁰ ffu/ml in both tissue culture flasks and bioreactors.

Gene Deletion and Site-Directed Mutagenesis in the Reverse GeneticsSystem

Deletion of the Psi Region of the Rabies Virus ERA Genome

The complete Psi-region of the rabies virus ERA genome was deleted asfollows: 3′Δψ fragment was amplified using pTMF as template by PCR withprimers (5Δψ: CCCTCTGCAGTTTGGTACCGTCGAGAAAAAAACATTAGATCAGAAG; SEQ ID NO:51, Pst1 and Kpn1 recognition sites underlined; and Le3-Blp primer) andwas cloned to pCR-BluntII-TOPO vector (Invitrogen, Carlsbad, Calif.) forthe construction of pPΔ5ψ plasmid. The 5′Δψ fragment was amplified usingthe same template by PCR with primers (SnaB5: ATGAACTTTCTACGTAAGATAGTG;SEQ ID NO: 52, SnaB1 recognition site underlined; and 3Δψ:CAAACTGCAGAGGGGTGTTAGTTTTTTTCAAAAAGAACCCCCCAAG; SEQ ID NO: 53, Pst1recognition site underlined) was successively cloned to the above pPΔ5ψplasmid to finish the construction of the pPΔΨ plasmid. The fragmentrecovered by SnaB1 and Pst1 restriction enzyme digestion from the pPΔψplasmid substituted the counterpart in the pSKF construct to make thepSKFΔψ plasmid. The whole DNA fragment containing the ERA genomic cDNA,digested by Nhe1 and Not1 from pSKFΔψ plasmid, was re-cloned to thepcDNA3.1/Neo (+) plasmid to finalize the construction of pTMFΔψ. Forverification of the rescued strain lacking Psi, designated Era-, primerscovering the Psi-region were applied in RT-PCR with total RNA fromERA-infected BSR cells. A 400 bp fragment corresponding to the Psiregion was amplified only from rERA virus, but not from ERA. Sequencedata verified the complete deletion of the Psi-region.

Deletion of the Glycoprotein Gene in the Rabies Virus ERA Genome:

The 5′gΔψ fragment was amplified using pSKF as template by PCR withprimers (SnaB5 primer, and 3Δg:CAAACTGCAGAGGGGTGTTAGTTTTTTTCACATCCAAGAGGATC; SEQ ID NO: 54). Afterdigestion by SnaB1 and Pst1 restriction enzymes, this recovered fragmentwas cloned to replace its counterpart in the pSKFΔψ construct. The 3′gΔψfragment was amplified using the same template by PCR with primers (5Δg:CCTCTGCAGTTTGGTACCTTGAAAAAAACCTGGGTTCAATAG; SEQ ID NO: 55, and Le3-Blpprimer), and was consecutively cloned to the modified pSKFΔψ, to replaceits counterpart. The final fragment, recovered by SnaB1 and Blp1restriction enzymes cut from this pSKFΔψ without the G gene, wasre-cloned to pcDNA3.1/Neo (+) plasmid to form the pTMFΔg construct forvirus recovery.

Glycoprotein Gene Site-Directed Mutagenesis:

Site directed mutagenesis to introduce a three nucleotide change fromAGA to GAG at amino acid position 333 of the glycoprotein was performedas previously described (Wu et al., J. Virol. 76: 4153-4161, 2002). Theprimers in the mutagenesis reaction were M5G primer:CTCACTACAAGTCAGTCGAGACTTGGAATGAGATC (SEQ ID NO: 56, the three mutatednucleotides in bold) and M3G primer: GACTGACTTTGAGTGAGCATCGGCTTCCATCAAGG(SEQ ID NO: 57). For the recovered strain (ERAg3), three nucleotidechanges from AGA to GAG at amino acid position 333 (aa333) wereconfirmed by sequencing after RT-PCR with primers 5G and 3G. Afterconfirmation by DNA sequencing, the mutated G was cloned back to thepTMF plasmid to make the pTMFg3 construct for virus recovery. Theglycoprotein encoded by this mutated G gene is represented by SEQ ID NO:58.

Incorporation of an Exogenous ORF into ERA Rabies Virus Genome

To express exogenous ORFs in RV, an extra transcription unit with Pst1and Kpn1 recognition sites were created and incorporated at the Psi orP-M gene intergenic regions, respectively. In brief, for creation of anextra transcription unit at the Psi-region, the same steps werefollowed, except for the 5′Δψ fragment amplification step, the 3Δψprimer was changed to 3Δψcis:CCAAACTGCAGCGAAAGGAGGGGTGTTAGTTTTTTTCATGATGAACCCCCC AAGGGGAGG (SEQ IDNO: 59). The final construct without the Psi-region, but with an extratranscription unit, was designated as pMTFΔψcis. The GFP, ERA G, ormutated G at amino acid residues 333 were cloned to this transcriptionalunit to form pMTFgfp1, pMTF2g, pMTFg3, pMTF2g3 constructs, respectively,for virus rescue.

To incorporate an extra transcription unit to the P-M intergenic region,the cisp5 fragment was amplified using pMTF as template with primerscis55: GACTCACTATAGGGAGACCCAAGCTGGCTAGCTGTTAAG (SEQ ID NO: 60), cis53:CCAAACTGCAGCGAAAGGAGGGGTGTTAGTTTTTTTCATGTTGACTTTAGGA CATCTCGG (SEQ IDNO: 61), and was cloned in substitution of its counterpart in the pMTFplasmid. The cisp3 fragment was amplified and cloned in a similar waywith primers cis35: CCTTTCGCTGCAGTTTGGTACCGTCGAGAAAAAAACAGGCAACACCACTGATAAAATGAAC (SEQ ID NO: 62) and cis33: CCTCCCCTTCAAGAGGGCCCCTGGAATCAG(SEQ ID NO: 63). After assembling the cisp5 and cisp3 fragmentstogether, the final construct was designated as pMTFcisp, for acceptingORFs. The recombinant construct containing the GFP gene was namedpTMFgfp2 for virus recovery.

To produce an ERA derivative, designated ERAgm, in which theglycoprotein encoding sequence was reversed in order with the matrixprotein encoding sequence, the glycoprotein gene was deleted asdescribed above. The G gene (amplified as disclosed above) was theninserted between P and M genes, yielding a rabies virus genome in theorder of N-P-G-M-L. Similarly, the same strategy was applied to producethe ERAg3m derivative, in which the glycoprotein has a triple nucleotidemutation at 333 amino acid residue (from AGA to GAG) by substituting theG gene produced by site directed mutagenesis as described above. Toproduce the ERAgmg construct, an extra copy of glycoprotein gene wasinserted between P and M genes, and made the rabies virus genome in theorder of N-P-G-M-G-L.

An extra transcription unit was modified and incorporated into twodifferent regions of the ERA genome, namely psi-region and P-Mintergenic region. When heterologous ORFs are incorporated into thesetranscription units, designated trans 1 and trans 2, respectively,efficient production of the encoded product results. Sequence of thetranscription unit is: CTAACACCCCTCCTTTCGCTGCAGTTTGGTACCGTCGAGAAAAAAA(SEQ ID NO: 64, Pst1 and Kpn1 were underlined).

Example 6 Recovery of Parental and Derivative Viruses

This example describes the recovery of parental ERA virus and exemplaryderivatives using the reverse genetics system disclosed herein. BSRcells were transfected at near 80% confluence in six-well-plates withviral full length transcription plasmid pTMF (pTMFΔψ, pTMFg3, pTMF2g,pTMF2g3, pTMFgfp1, pTMFgfp2, pTMFΔg, pTMFgm, or pTMFgmg, respectively)at 3 μg/well, together with five helper plasmids: pTN (1 μg/well), pMP(0.5 μg/well), pML (0.5 μg/well), pMG (0.5 μg/well) and pNLST7 (1μg/well) by TransIT-LT1 reagent (Mirus, Madison, Wis.) following theprotocol recommended by the manufacturer. Four days after transfection,1 ml of fresh BSR cell suspension (about 5×10⁵ cells) was added to eachwell. Cells were incubated at 37° C., 5% CO₂ for 3 days. Cellsupernatants were collected for virus titration.

To titrate the recovered virus, monolayers of BSR cells in LAB-TEKeight-well-plates (Naperville, Ill.) were infected with serial 10-folddilutions of virus supernatant and incubated at 37° C., 0.5% CO₂ for 48h. Cells were fixed in 80% chilled acetone at room temperature for 1 hand stained with FITC-labeled anti-rabies virus N monoclonal antibody at37° C. for 30 minutes. After three rinses of the plates with PBS,stained foci were counted using direct fluorescent microscopy. Detailsfor direct RV fluorescent assay (DFA) can be found on the World Wide Webatcdc.gov/ncidod/dvrd/rabies/professional/publications/DFA-diagnosis/DFA_protocol.htm.

All of the viruses except ERA-G were recovered at high titer fromcultured BSR cells as indicated in Table 3. Surprisingly, rearrangementand switching of the G gene with the M gene did not hinder recovery ofrecombinant derivative ERA virus. Rearrangement of the G gene in the RVgenomes was previously not believed feasible due to cell death fromoverexpression of G protein (Faber et al., J. Virol. 76:3374-3381,2002). However, these results demonstrate that rearrangement is possiblein the ERA strain. Accordingly, it is likely that RV gene shuffling ispossible not only for the G gene, but also for other genes as well.

The ERA-G (without G) virus was recovered after plasmid transfectionfollowing the same procedure as for the other viral constructs rescue,but virus foci were very limited and restrained in local areas after thefirst round of transfection. The rescued virus was not capable ofspreading further to the nearby healthy BSR cells (FIG. 4A) even afterone week of incubation at 37° C., 5% CO₂. Infection of normal BSR cellswith the above transfection supernatants presented single cell stainingin the DFA test, which suggested the recovered virus was incapable ofspread. To amplify the ERA-G virus, a BHK cell line constitutivelyexpressing ERA G was established as described in Example 4 (designatedBHK-G). By indirect fluorescent assay screening, a pool of BHK cellsexpressing G were selected and maintained for amplification of ERA-Gvirus (FIG. 4B). With the aid of the BHK-G cell line, ERA-G virus grewto 10⁷ ffu/ml. Total RNA from ERA-G virus-infected BHK-G cells wasextracted for Northern blot analysis (FIG. 4C) with a G gene probe. TheG gene was absent in the viral genomic RNA, however G mRNA was detected,which came from infected supportive BHK-G cells. In purified ERA-G viralgenomic RNA, no hybridization signal was detected by G probe, indicatingthe deletion of the G gene in the ERA genome.

Example 7 Growth of Rescued ERA Virus and its Derivatives to High Titerin a Bioreactor

In oral vaccine development, high virus titer is typically required toelicit reliable immunity after administration. This example demonstratesthat the ERA virus and derivatives can be grown to high titer in abioreactor at volumes applicable to commercial scale-up. All 10 rescuedERA viruses were amplified in a bioreactor, CELLine AD1000 (IBS IntegraBioscience, Chur, Switzerland) to titers ranging from 10⁷ to 10¹⁰ffu/ml. In brief, BSR cells were transfected with the exemplaryantigenome transcription vectors and helper vectors, as described above.Cells were inoculated at a multiplicity of infection of 1 virion percell, at a concentration of 10⁶ cells/ml in one tenth the bioreactorvessel volume. Transfected cells were grown at 37° C., 5% Co₂ in DMEMsupplemented with 10% fetal bovine serum. The supernatant was harvestedevery three to five days for between two and three harvests. Thedeficient ERA-G grew less well compared with other viruses, with only10⁸ ffu/ml for the ERA-G (TABLE 3. and FIG. 5).

TABLE 3 Full-length plasmid constructs and corresponding rescued virusesPlasmid Rescued Titers ffu/ml from Titers ffu/ml constructs virusescultured cells in bioreactors pTMF rERA  5 × 10⁷   3 × 10¹⁰ pTMFΔψ ERA-6.3 × 10⁷  3.2 × 10¹⁰ pTMFg3 ERAg3  3 × 10⁶ 1.8 × 10⁹ pTMFgfp1 ERAgreen13.5 × 10⁶ 5.6 × 10⁹ pTMFgfp2 ERAgreen2  2 × 10⁷ 6.2 × 10⁹ pTMF2g ERA2g1.6 × 10⁶ 3.9 × 10⁹ pTMF2g3 ERA2g3  8 × 10⁷ 4.6 × 10⁹ pTMFΔg ERA-G 1.2 ×10² 1.5 × 10⁷ pTMFgm ERAgm 5.31 × 10⁶  1.9 × 10⁹ pTMFgmg ERAgmg 3.1 ×10⁶  1.2 × 109

Example 8 Expression of Exogenous Proteins from Extra TranscriptionalUnits in Rabies Virus

This example demonstrates the expression of recombinant proteins from aheterologous ORF inserted into a rabies virus vector. In this example,the ERA virus vector is used as a prototype rabies virus vector. Toconstruct ERA virus as a vector for accepting ORFs, a conservative RVtranscriptional unit between the N and P genes was modified andintroduced into the ERA genome at two different locations: 1) at the psiregion (trans 1), and 2) at the P-M intergenic region (trans 2). Thetranscriptional unit was designed to possess two unique restrictionenzyme recognition sites to facilitate introduction of heterologouspolynucleotide sequences:(TTTTTTTGATTGTGGGGAGGAAAGCGACGTCAAACCATGGCAGCTCTTTTTT T: SEQ ID NO: 65,Pst1 and Kpn1 sites underlined).

In a first example, the GFP gene was cloned into this unit for virusrecovery, since GFP expression could be observed directly under a UVmicroscope when the transfected BSR cells were still incubating.Expression of the GFP protein was directly visible by fluorescentmicroscopy with an excitation filter of 470±20 nm. The ERAgreen2 (GFPgene inserted after P gene in RV genome-trans 2)-infected cells showedclear green foci after three days of plasmid transfection, whileERAgreen1 (GFP gene inserted after G gene in the “traditional” Ψregion-trans 1) did not present obvious green foci until five dayspost-transfection (FIG. 6). The introduced transcriptional unit wasfunctional in the RV genome at both locations, although expression andaccumulation was apparent more rapidly when GFP was expressed from trans2. Thus, these results also indicate that the level of expression from aheterologous ORF can be modulated by selecting the transcription unitinto which the ORF is cloned.

In other examples, 1) an additional copy of ERA G; or 2) an additionalcopy of ERA G with an amino acid substitution at position 333, wasincorporated into the ERA viral genome. The successfully rescued viruseswere named ERA2g and ERA2g3, respectively. Since quantitation of viral Gexpression was not practical, the relative increase in expression levelsof G in ERA2g and ERA2g3-infected cells was confirmed by Northern-blotwith a G probe. In brief, the ERA G gene probe was labeled using the DigDNA Labeling Kit (Roche, Indianapolis, Ind.) and imaged with Dig NucleicAcid Detection Kit (Roche, Indianapolis, Ind.) and was measured bydensity spectrophotometry (FIG. 7). The tandem linked G genes in therecovered viruses were also confirmed by RT-PCR with 5G and 3G primers.A predominant band indicating a single G copy was observed at 1.5 kb. Inaddition, a second weaker band was observed at approximately 3.0 kbindicative of the two Gs in a tandem arrangement.

These results demonstrate that introduction of transcription units intothe ERA genome can be used to express diverse heterologous proteins fromintroduced ORFs. Furthermore, expression of the protein encoded by theheterologous ORF is modulated by the position into which the ORF isinserted. Thus, ERA virus is a widely adaptable vector for theexpression of recombinant proteins.

Example 9 In Vivo Immune Response to Engineered Viruses

This example demonstrates the in vivo effects of inoculation with theengineered ERA virus and exemplary derivatives. All animal care andexperimental procedures were performed in compliance with the CDCInstitutional Animal Care and Use Guidelines. Eighty three-week old micewere divided into 8 groups of 10 each for intramuscular (i.m)administration of recovered viruses (10⁶ ffu of virus per mouse). Tenhealthy mice were held as uninfected mock controls. For the ERA andERAg3 constructs, additional intracerebral (i.c) injections of the samedose of viruses were applied to another group of ten three-week oldmice. In two-day old suckling mice, only the ERAg3 and ERA-G viruseswere inoculated intracerebrally, with the same dose. Animals werechecked daily for illness. Ill animals were euthanized by CO₂intoxication and brains were removed for rabies virus diagnosis. Tendays after infection, blood was collected by the retro orbital route andsera obtained for neutralizing antibody assays, following the standardrapid fluorescent focus inhibition test (RFFIT) (Smith et al., Bulletinof the World Health Organization. 48: 535-541, 1973). One month afterinfection, surviving animals were challenged with a lethal dose ofstreet rabies virus (dog/coyote salivary gland homogenate) (Orciari etal., Vaccine. 19:4511-4518, 2001).

Mouse monoclonal antibody (Mab 523-11) against rabies virus G wasmaintained at CDC (Hamir et al., Vet Rec. 136, 295-296, 1995) andFITC-conjugated anti-N monoclonal antibody was purchased from Centocor(Horsham, Pa.). T7 RNA polymerase monoclonal antibody was from Novagen(Madison, Wis.). Goat anti-mouse IgG-FITC conjugate was purchased fromSigma-Aldrich (St. Louis, Mo.). Anti-rabies virus G monoclonal antibody(Mab 1-909) was maintained at CDC and goat anti-human IgG-FITC conjugatewas purchased from Sigma-Aldrich (St. Louis, Mo.).

Among the three-week old mice inoculated intramuscularly by the eightdifferent virus constructs, 50% of mice inoculated with ERA (rERA) orERA-, and 20% of mice inoculated with ERAgreen1 showed mild neurologicalsigns at 10 days after inoculation. No other groups showed any signsuggestive of rabies virus infection (FIG. 8A). Sera were collected forneutralizing antibody titration before challenge. The ERA2g (5.60 IU)and ERA2g3 (5.61 IU) elicited higher titers than the single-copy G virusconstructs (FIG. 8E). Mice surviving one month after inoculation weresubjected to challenge with a lethal dog/coyote street virus (0.05 ml,maintained at CDC for standard animal challenge tests). In the ERA andERA-groups, 40 to 62% of the mice showed mild rabies signs,respectively, and were euthanized. All other groups survived without anysigns of rabies (FIG. 8B). In the i.c groups, three-week old micesurvived after ERAg3 inoculation, but succumbed after ERA injection(FIG. 8C). The ERA-G construct did not kill 2-day old suckling mouse,however ERAg3 was virulent enough to kill all infected suckling mice(FIG. 8D). Exemplary antibody titers are shown in Table 4.

TABLE 4 Production of rabies specific antibodies Group Average Titer ERA433 G333 468 2G 560 2G333 561 -PSI 490 GFP 437 G green 833 G minus 136Controls <⅕

These data demonstrate that all of the ERA based viruses were capable ofeliciting an immune response following inoculation. As expected, theparental ERA virus was virulent, resulting in substantial morbidity andmortality in infected animals. In contrast, the various exemplaryderivatives elicited a protective immune response when mice wereinoculated prior to challenge.

In addition to the pre-exposure assessment described above, the abilityof the ERA virus derivatives to elicit a protective immune responsefollowing infection with virulent rabies virus was determined. In brief,groups of hamsters were infected with one of three different strains ofrabies virus (n=9 per group), and either given the recombinant vaccine(ERA-g333), or rabies immune globulin plus inactivated commercial rabiesvaccines. Approximately 80-100% of control animals succumb, whereasapproximately 60-100% of vaccinated animals survive as shown in FIGS.9A-C. These results demonstrate that post-exposure administration of thederivative rabies virus confers substantial protection against differentstrains of rabies virus.

In view of the many possible embodiments to which the principles of thedisclosed invention may be applied, it will be recognized that theillustrated embodiments are only preferred examples of the invention andshould not be taken as limiting the scope of the invention. Rather, thescope of the invention is defined by the following claims. We thereforeclaim as our invention all that comes within the scope and spirit ofthese claims.

The invention claimed is:
 1. A vector system comprising: a first vectorcomprising a full-length rabies virus antigenomic DNA, wherein thefull-length antigenomic DNA comprises the nucleic acid sequence of SEQID NO: 17; and, a plurality of helper vectors comprising nucleic acidsthat encode at least one rabies virus strain ERA protein, whereinexpression of the plurality of vectors in a transfected host cellresults in production of a recombinant rabies virus.
 2. The vectorsystem of claim 1, wherein the first vector comprises in a 5′ to 3′direction: a hammerhead ribozyme; a rabies virus antigenomic DNAcomprising the nucleic acid sequence of SEQ ID NO: 17; and a hepatitisdelta virus ribozyme, wherein a plurality of nucleotides of thehammerhead ribozyme are complementary to the antisense genomic sequenceof the rabies virus.
 3. The vector system of claim 2, whereintranscription of the antigenomic DNA is under the transcriptionregulatory control of at least one of the CMV promoter and the phage T7RNA polymerase promoter.
 4. The vector system of claim 1, wherein theplurality of helper vectors comprises: a vector comprising apolynucleotide sequence that encodes a rabies virus N protein; a vectorcomprising a polynucleotide sequence that encodes a rabies virus Pprotein; a vector comprising a polynucleotide sequence that encodes arabies virus M protein; a vector comprising a polynucleotide sequencethat encodes a rabies virus L protein; and a vector comprising apolynucleotide sequence that encodes a phage T7 RNA polymerase.
 5. Thevector system of claim 4, further comprising a vector comprising apolynucleotide sequence that encodes a rabies virus G protein.
 6. Thevector system of claim 5, wherein transcription of one or more of thepolynucleotide sequences that encode the rabies virus P, M, L or Gprotein or the T7 polymerase are under the transcription regulatorycontrol of both the CMV promoter and the T7 promoter.
 7. A recombinantvirus genome comprising the nucleic acid sequence as set forth in SEQ IDNO:
 17. 8. The recombinant virus genome of claim 7, further comprising avector.
 9. A recombinant virus comprising a genome as set forth in claim7.
 10. A live rabies virus vaccine comprising at least one recombinantrabies virus genome, wherein the at least one recombinant rabies virusgenome comprises the sequence shown in SEQ ID NO:
 17. 11. A method ofproducing a live rabies virus vaccine, comprising introducing the vectorsystem of claim 4 into a host cell, and recovering live recombinantrabies virus.
 12. A method of vaccinating a subject against rabies,comprising administering an effective amount of the live rabies virusvaccine produced according to the method of claim 11 to a subject, suchthat cells of the subject are infected with the rabies virus vaccine,wherein an anti-rabies immune response is produced in the subject. 13.The method of claim 12, wherein the subject is a human.
 14. The methodof claim 12, wherein the subject is a non-human animal.
 15. The methodof claim 12, wherein the administration comprises oral administration.16. The method of claim 15, wherein the oral administration comprisesadministration through food-baits designed to vaccinate wild animalpopulations.
 17. A pharmaceutical composition comprising the live rabiesvaccine of claim 10 and a pharmaceutically acceptable carrier orexcipient.
 18. A method of eliciting an immune response against rabiesin a subject, comprising administering an effective amount of therecombinant rabies virus of claim 9 to a subject, such that cells of thesubject are infected with the rabies virus, wherein an anti-rabiesimmune response is produced in the subject.
 19. A method of producing arecombinant rabies virus, comprising introducing the vector system ofclaim 4 into a host cell, and recovering recombinant rabies virus.
 20. Amethod of producing a recombinant rabies virus, comprising introducingthe vector system of claim 5 into a host cell, and recoveringrecombinant rabies virus.